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US20030158382A1 - Novel collectins - Google Patents

Novel collectins Download PDF

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Publication number
US20030158382A1
US20030158382A1 US10/258,105 US25810503A US2003158382A1 US 20030158382 A1 US20030158382 A1 US 20030158382A1 US 25810503 A US25810503 A US 25810503A US 2003158382 A1 US2003158382 A1 US 2003158382A1
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gly
amino acid
seq
pro
set out
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US10/258,105
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Nobutaka Wakamiya
Hiroyuki Keshi
Katsuki Ohtani
Takashi Sakamoto
Yuichiro Kishi
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Fuso Pharmaceutical Industries Ltd
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Fuso Pharmaceutical Industries Ltd
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Assigned to FUSO PHARMACEUTICAL INDUSTRIES reassignment FUSO PHARMACEUTICAL INDUSTRIES ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KESHI, HIROYUKI, KISHI, YUICHIRO, OHTANI, KATSUKI, SAKAMOTO, TAKASHI, WAKAMIYA, NOBUTAKA
Publication of US20030158382A1 publication Critical patent/US20030158382A1/en
Priority to US12/334,052 priority Critical patent/US7985833B2/en
Priority to US13/087,989 priority patent/US20120015400A1/en
Priority to US13/160,091 priority patent/US8604162B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to isolated human and mouse collectins (herein referred to as “hCL-L2” and “mCL-L2” respectively, or merely as “CL-L2” when discrimination of both is not intended), genes and proteins, the homologues, mutants, modified forms and polymorphic variants thereof (these are collectively referred to as “derivatives”), fragments thereof (hereinafter collectively referred to as “CL-L2s” for all of these), and the detection of the same.
  • the present invention further relates to compositions which comprise CL-L2s for pharmaceutical use, diagnostic use and research use, and methods for the production and use of the same.
  • the present invention relates to agonists and antagonists of CL-L2s proteins, as well as methods for screening drugs using CL-L2s. Moreover, the present invention also relates to expression vectors comprising CL-L2s gene, transformed cells that were transformed with the expression vector, antibodies to CL-L2s protein, and cells that produce the antibody.
  • Complement systems that play an important role in a body defense mechanism are known to include: a classic pathway in which an immunoglobulin serves as a recognition molecule followed by the activation of C1 that is the first component of the complement; and an alternative pathway in which C3, which is the third component of the complement, is directly coupled to foreign substances such as bacteria.
  • a lectin pathway was illustrated in which a mannose binding protein (hereinafter referred to as “MBP”), which is a serum lectin, activates the complement system through the direct recognition of and coupling with a carbohydrate chain on the surface of the foreign substance, in recent years (Sato, T. et al., Int. Immunol., 6, 665-669, 1994).
  • MBP mannose binding protein
  • MBP is a C type lectin which specifically binds to mannose, N-acetylglucosamine and the like in the presence of Ca, of which structure comprises a collagen-like domain containing at least one (Gly-Xaa-Yaa)n, and carbohydrate chain recognition domain (CRD).
  • lectins having a collagen-like domain and CRD are generically called as collectin (Malhotora, R. et al., Eur. J.
  • Collectin has an opsonic activity, which is believed to participate in fundamental immunity against a variety of microorganisms such as bacteria and viruses (Kawasaki, N. et al., J. Biochem., 106, 483-489, 1989; Ikeda, K. et al., J. Biol. Chem., 262, 7451-7454, 1987; Ohta, M. et al., J. Biol. Chem., 265, 1980-1984, 1990; Summerfield, J. A. et al., Lancet, 345, 886, 1995).
  • These collectins are known to be constituted from a basic structure containing characteristic domains such as (1) CRD and (2) collagen-like domain and the like as shown in FIG. 1( a ) (Malhortra et al., Eur. J. Immunol., 22, 1437-1445, 1992).
  • This basic structure forms a subunit through composing a triple helix at the collagen-like domain, and thus these subunits further form an oligomer structure such as trimer, tetramer, hexamer and the like.
  • BKg and MBP inhibit infections by H1 and H3 types influenzae A viruses as well as a haemagglutination activity (Wakamiya et al., Glycoconjugate J., 8, 235, 1991; Wakamiya et al., Biochem. Biophys. Res. Comm., 187, 1270-1278, 1992). Thereafter, a cDNA clone encoding BKg was obtained, and the relevance between BKg and SP-D and the like has been also found (Suzuki et al., Biochem. Biophys. Res. Comm., 191, 335-342, 1993).
  • collectins are substances to which usefulness in the elucidation of body defense mechanisms and utilities as a biologically active substance are expected.
  • the finding of novel molecular species belonging to this family may greatly contribute in various medical fields and biological fields in addition to the therapy of infectious diseases.
  • the object of the present invention is to provide materials that can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and for the development of reagents and drugs for the same.
  • a protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 2, or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 45 (corresponding to a base sequence set out in the base position 265-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2;
  • a protein comprising an amino acid sequence consisting of 245 amino acids set out in the amino acid position 1-245 of SEQ ID NO: 4, or a protein consisting of an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 46 (corresponding to a base sequence set out in the base position 141-875 of SEQ ID NO: 3), a base sequence encoding an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4;
  • a protein comprising an amino acid sequence consisting of 159 amino acids set out in SEQ ID NO: 47 (corresponding to an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2), or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 48 (corresponding to a base sequence set out in the base position 601-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2;
  • the protein further comprising a sequence (Gly-Xaa-Yaa)n, wherein n represents an integer of from 1 to 50, Xaa and Yaa represent an amino acid residue, which may be the same or different, at an N-terminal of the protein according to (5);
  • a protein comprising an amino acid sequence of:
  • a protein comprising a sequence of:
  • a protein comprising an amino acid sequence consisting of 197 amino acids set out in the amino acid position 1-197 of SEQ ID NO: 6, or a protein consisting of an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO:55 (corresponding to a base sequence set out in the base position 141-731 of SEQ ID NO: 5), a base sequence encoding an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6;
  • a protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 8, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 56 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 7), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8;
  • a protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 10, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 57 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 9), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10;
  • a protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 13, or a protein consisting of an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 58 (corresponding to a base sequence set out in the base position 157-969 of SEQ ID NO: 12), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13;
  • a protein comprising an amino acid sequence consisting of 223 amino acids set out in the amino acid position 1-223 of SEQ ID NO: 37, or a protein consisting of an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 59 (corresponding to a base sequence set out in the base position 265-933 of SEQ ID NO: 36), a base sequence encoding an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37;
  • a protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 39, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 60 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 38), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39;
  • a protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 41, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41, or a derivative or a fragment thereof;
  • a base sequence set out in SEQ ID NO: 61 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 40), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in amino acid position 1-247 of SEQ ID NO: 41;
  • a process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to (2), (4), (6), (9), (12), (14), (16), (18), (22), (24) or (26); and harvesting thus produced hCL-L2s protein;
  • a process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to (20); and harvesting thus produced mCL-L2s protein;
  • the antibody according to (34) which is a polyclonal antibody, a monoclonal antibody or a peptide antibody;
  • FIG. 1 is a schematic drawing illustrating a basic structure (a) of principal collectins reported heretofore and an overview of the protein (MBP, SP-A and SP-D).
  • FIG. 2 is a drawing illustrating a preceding half of an alignment of amino acid sequences of four kinds of collectins reported heretofore.
  • FIG. 3 is a drawing illustrating a latter half of the alignment to that of FIG. 2.
  • FIG. 4 depicts drawings (a) and (b) showing each primer employed for the determination of the sequence of the novel collectin of the present invention, and the novel collectin obtained thereby.
  • FIG. 5 is a drawing illustrating a preceding half of an alignment of amino acid sequences of four kinds of collectins reported heretofore and the novel collectin (hCL-L2-1) of the present invention.
  • FIG. 6 is a drawing illustrating a latter half of the alignment to that of FIG. 5.
  • FIG. 7 shows results of analysis of distribution of mRNA in various human tissues demonstrating the tissue distribution of the novel collectin hCL-L2 of the present invention.
  • FIG. 8 shows a phylogenetic tree of various collecting.
  • FIG. 9 shows results of analysis of distribution of mRNA in various human tissues demonstrating the tissue distribution of the novel collectin hCL-L2-1 and hCL-L2-2 of the present invention.
  • FIG. 10 is a schematic drawing illustrating a sugar binding characteristics of the novel collectin hCL-L2-1 of the present invention.
  • the present inventors successfully cloned human and mouse novel collectin genes (hCL-L2-1 and mCL-L2).
  • a CRD amino acid position 113-271 of SEQ ID NOs: 2 and 13, amino acid position 87-245 of SEQ ID NO: 4
  • a collagen-like domain the amino acid position 41-112 of SEQ (ID NOs: 2 and 13, the amino acid position 18-86 of SEQ ID NO: 4) having a sequence of (Gly-Xaa-Yaa)n were present at C-terminal of the novel CL-L2.
  • N-terminal amino acids of CL-L2-1 position 1-43 set out in SEQ ID NO: 2
  • N-terminal amino acids of CL-L2-2 position 1-17 set out in SEQ ID NO: 4 were different, whilst the rest being identical.
  • position of 1-43 of N-terminal amino acid of CL-L2-1 contained a signal sequence and one coil of a collagen-domain and the like, there were neither a signal sequence nor one coil of a collagen-like domain in position of 1-17 of N-terminal amino acid of CL-L2-2.
  • CL-L2-2v1 SEQ ID NO: 5, 6
  • CL-L2-2v2 SEQ ID NO: 7, 8
  • CL-L2-2v3 SEQ ID NO: 9, 10
  • CL-L2-2v1 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 18 to 65 (i.e., deletion of the base position of from 192 to 335 of CL-L2-2 set out in SEQ ID NO: 3);
  • CL-L2-2v2 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 18 to 41 (i.e., deletion of the base position of from 192 to 263 of CL-L2-2 set out in SEQ ID NO: 3);
  • CL-L2-2v3 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 42 to 65 (i.e., deletion of the base position of from 264 to 335 of CL-L2-2 set out in SEQ ID NO: 3).
  • CL-L2-1v1 SEQ ID NO: 36, 37
  • CL-L2-1v2 SEQ ID NO: 38, 39
  • CL-L2-1v3 SEQ ID NO: 40, 41
  • CL-L2-1v1 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino acid position of from 44 to 91 (i.e., deletion of the base position of from 394 to 537 of CL-L2-1 set out in SEQ ID NO: 1);
  • CL-L2-1v2 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino acid position of from 44 to 67 (i.e., deletion of the base position of from 394 to 465 of CL-L2-1 set out in SEQ ID NO: 1);
  • CL-L2-1v3 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino-acid position of from 68 to 91 (i.e., deletion of the base position of from 466 to 537 of CL-L2-1 set out in SEQ ID NO: 1).
  • hCL-L2 gene used herein includes hCL-L2-1 set out in SEQ ID NO: 1, hCL-L2-2 set out in SEQ ID NO: 3, hCL-L2-2v1 set out in SEQ ID NO: 5, hCL-L2-2v2 set out in SEQ ID NO: 7, hCL-L2-2v3 set out in SEQ ID NO: 9, hCL-L2-1v1 set out in SEQ ID NO: 36, hCL-L2-1v2 set out in SEQ ID NO: 38, and hCL-L2-1v3 set out in SEQ ID NO: 40 respectively, unless otherwise stated.
  • hCL-L2 protein used herein includes hCL-L2-1 set out in SEQ ID NO: 2, hCL-L2-2 set out in SEQ ID NO: 4, hCL-L2-2v1 set out in SEQ ID NO: 6, hCL-L2-2v2 set out in SEQ ID NO: 8, hCL-L2-2v3 set out in SEQ ID NO: 10, hCL-L2-1v1 set out in SEQ ID NO: 37, hCL-L2-1v2 set out in SEQ ID NO: 39, and hCL-L2-1v3 set out in SEQ ID NO: 41 respectively, unless otherwise stated.
  • homologues, variants, modified forms and polymorphic variants thereof are also included. These may be naturally occurring or artificially produced.
  • derivatives include any of the foregoing substances.
  • mCL-L2 gene used herein includes mCL-L2 set out in SEQ ID NO: 12, unless otherwise stated.
  • mCL-L2 protein used herein includes mCL-L2 set out in SEQ ID NO: 13, unless otherwise stated.
  • homologues, variants, modified forms and polymorphic variants thereof (these are collectively referred to as “derivatives”), and fragments of the same are also included. These may be naturally occurring or artificially produced.
  • the present invention includes any of the foregoing substances.
  • the present invention also involves amino acid sequences substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13; and base sequences encoding amino acid sequences substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13.
  • amino acid sequence substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13 refers to the amino acid sequence having alteration such as substitution, deletion, addition and/or insertion of one or several amino acids therein as long as the protein has an equal property to that of the protein comprising an amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13.
  • SEQ ID NO: 47 corresponding to the amino acid position of 113-271 of SEQ ID NO: 2
  • amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13 may be naturally occurring, or artificially
  • Such deletion, substitution, and/or addition of one or several amino acids referred to herein may be deletion, substitution, and/or addition of amino acid(s) without large extent of alteration of residues having hydrophobic, hydrophobic, acidic, basic group or the like of the novel collectin, and with small extent of alteration of each fundamental characteristics of (1) Ca 2+ dependent carbohydrate chain recognition domain (CRD) and (2) collagen-like domain.
  • CCD Ca 2+ dependent carbohydrate chain recognition domain
  • deletion, substitution, and/or addition of for example, (1) from about 1 to 10 amino acids in Ca 2+ dependent carbohydrate chain recognition domain (CRD) and (2) from about 1 to 50, preferably from about 1 to 15 amino acids in collagen-like domain may be allowed.
  • Equal property herein refers to a property inherent to a protein having an amino acid sequence prior to the alteration, e.g., a feature involving in tertiary structure of a protein.
  • the present invention also involves a nucleic acid sequence set out in SEQ ID NO: 1, 3, 5, 7, 9, 12, 36, 39 or 41, SEQ ID NO: 48 (corresponding to the base position of 601-1077 of SEQ ID NO: 1) or in the nucleic acid position of 493-969 of SEQ ID NO: 12, or a nucleic acid sequence comprising a fragment thereof, or a nucleic acid sequence that can hybridize to a nucleic acid sequence complementary thereto (hereinafter, referred to as “specified sequence”) under a stringent condition.
  • the stringent condition according to the present invention may involve a condition for example; incubating in a solution containing 5 ⁇ SSC, 5% Denhardt's solution (0.1% BSA, 0.1% Ficol 1400, 0.1% PVP), 0.5% SDS and 20 ⁇ g/ml denatured sermon sperm DNA at 37° C. overnight followed by a wash with 2 ⁇ SSC containing 0.1% SDS at room temperature.
  • SSPE may be employed in place of SSC.
  • resultant nucleic acid sequence is speculated to exhibit at least 50% or greater homology to the specified sequence.
  • Many of the proteins encoded by a nucleic acid sequence that can hybridize to the specified sequence under a stringent hybridization condition are believed to have an equal property to CL-L2s protein. Therefore, such proteins are also involved in the present invention as long as they have an equal property to CL-L2s protein.
  • amino acid sequence of hCL-L2-1 set out in SEQ ID NO: 2 represents a protein consisting of 271 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 45) consists of 813 bases. Characteristic amino acid sequences such as those of a signal sequence, a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 1.
  • amino acid sequence of hCL-L2-2 set out in SEQ ID NO: 4 represents a protein consisting of 245 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 46) consists of 735 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 3.
  • amino acid sequence of hCL-L2-2v1 set out in SEQ ID NO: 6 (amino acid position 1-197) represents a protein consisting of 197 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 55) consists of 591 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 5.
  • amino acid sequence of hCL-L2-2v2 set out in SEQ ID NO: 8 (amino acid position 1-221) represents a protein consisting of 221 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 56) consists of 663 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 7
  • amino acid sequence of hCL-L2-2v3 set out in SEQ ID NO: 10 (amino acid position 1-221) represents a protein consisting of 221 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 57) consists of 663 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 9.
  • amino acid sequence of hCL-L2-1v1 set out in SEQ ID NO: 37 (amino acid position 1-223) represents a protein consisting of 223 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 59) consists of 669 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 36.
  • amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 39 (amino acid position 1-247) represents a protein consisting of 247 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 60) consists of 741 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 38.
  • amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 41 (amino acid position 1-247) represents a protein consisting of 247 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 61) consists of 741 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 40.
  • amino acid sequence of mCL-L2 set out in SEQ ID NO: 13 represents a protein consisting of 271 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 58) consists of 813 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence.
  • the base sequence encoding this protein is set out in SEQ ID NO: 12.
  • Homologues used herein refer to nucleic acid sequences or amino acid sequences that bear high homology, which are homologous at least 50% or more, preferably 70% or more, more preferably 90% or more.
  • homologous search may be conducted which allows for gap junction.
  • the search may be performed using a procedure of multiple alignment (trade name: SODHO, Fujitsu Limited).
  • SODHO trade name: SODHO, Fujitsu Limited
  • algorithm for searching homology Smith-Waterman algorithm, which is the most accurate, may be employed.
  • FASTA or BLAST may be also utilized via the Internet.
  • Mutants used herein include for example, those resulting from allele, Single Nucleotide Polymorphism (SNP) and the like.
  • the nucleic acid sequence of the present invention may also include the mutated nucleic acid sequences derived from the changes in the range of degeneracy of the codon. Partial alteration of the codon of a nucleic acid sequence may be achieved according to a conventional procedure using such site directed mutagenesis methods as those in which a primer is employed consisting of a synthesized oligonucleotide that encodes the desired alteration (Mark, D. F. et al., Proc. Nati. Acad. Sci. USA., 81, 5662, 1984).
  • the mutated amino acid translated by the mutated codon has preferably similar properties to those of normal amino acid even in the case where the mutation is beyond the range of degeneracy of the codon.
  • the mutation may be preferably as follows, which are among amino acids having similar properties, functions, characteristics and the like, for example: the mutation among aliphatic amino acids such as alanine, valine, leucine and isoleucine; the mutation among neutral amino acids such as glycine, alanine, serine, threonine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, proline, tryptophan, asparagines and glutamine; the mutation among acidic amino acids such as aspartic acid and glutamic acid; the mutation among basic amino acids such as arginine, lysine and histidine; the mutation among serine and threonine, having a hydroxyl group; the mutation among phenylalanine and tyrosine, having an aromatic ring; and the like.
  • site-directed mutagenesis may be also included in the protein of the present invention.
  • site-directed mutagenesis may be
  • Modified forms used herein may be prepared using conventional techniques, for example, by acetylation, acylation, ADP-ribosylation, amidation, myristoylation, glycosylation, hydroxylation, phosphorylation, sulfation, formylation, methylation, polyethyleneglycolation, lipid coupling, nucleotide coupling, metal coupling (calcium addition and the like), fusion with other protein (albumin and the like), dimerization, and the like.
  • glycosylation does not occur when the host is in Escherichia coli , the expression may be conducted in eucaryotic cells when glycosylation is intended. Insect cells may be also used because glycosylation proceeds post-translationally in these cells, similarly to in mammalian cells.
  • Polymorphic variants used herein involve for example, polymorphisms caused by structural or conformational differences in chromosomal DNA, polymorphisms resulting from a change of a gene into its allelic gene, or the like.
  • genes of eucaryotic cells often exhibit polymorphic event, and this event may lead to the substitution of one or more amino acid(s), whilst the activity of the protein may be retained in spite of the substitution.
  • any of the genes encoding a protein obtained by artificially modifying the gene encoding any of the amino acid sequence set out in either SEQ ID NO: 2, 4, 6, 8, 10 or 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13 is involved in the present invention as far as the protein has a characteristic function of a gene according to the present invention.
  • any of the proteins prepared by artificial modification of amino acid sequence set out in either SEQ ID NO: 2, 4, 6, 8, 10 or 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13 is involved in the present invention as far as it has a characteristics of a protein according to the present invention.
  • the modification is construed as involving substitution, deletion, addition and/or insertion.
  • Fragments used herein refer to any optional fragments derived from the amino acid sequence of CL-L2s described above, which may include for example, an extracellular domain, an intracellular domain, a transmembrane domain, a collagen-like domain, a CRD domain, a collectin-like domain, a hydrophobic domain (a transmembrane domain and the like), a hydrophilic domain (domains other than hydrophobic domains), and the like, as well as fragments obtained by the fusion of these fragments.
  • exemplary fragments may include: a fragment comprising amino acids of approximately from the position 113 to 271 that form a CRD domain; a fragment comprising amino acids of approximately from the position 41 to 271 that form a CRD domain and a collagen-like domain; and a fragment comprising amino acids of approximately from the position 41 to 112 that form a collagen-like domain.
  • exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 245 that form a CRD domain and a collagen-like domain, and a fragment comprising amino acids of approximately from the position 18 to 86 that form a collagen-like domain; in the amino acid sequence of hCL-L2-2v1 set out in SEQ ID NO: 6, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 197 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 18 to 38 that form a collagen-like domain; in the amino acid sequence of hCL-L2-2v2 set out in SEQ ID NO: 8, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 221 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 18 to 62 that form a collagen-
  • exemplary fragments may include: a fragment comprising amino acids of from the position approximately 41 to 223 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 64 that form a collagen-like domain; in the amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 39, the fragments included may be: a fragment comprising amino acids of approximately from the position 41 to 247 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 88 that form a collagen-like domain; in the amino acid sequence of hCL-L2-1v3 set out in SEQ ID NO: 41, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 41 to 247 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 88 that
  • exemplary fragments may also include: a fragment comprising amino acids of approximately from the position 113 to 271 that form a CRD domain; a fragment comprising amino acids of approximately from the position 41 to 271 that form a CRD domain and a collagen-like domain; and a fragment comprising amino acids of approximately from the position 41 to 112 that form a collagen-like domain.
  • a CL-L2s gene according to the present invention may be those obtained through any processes.
  • the base sequence encoding CL-L2s of the present invention can be obtained by preparing mRNA from the cells that are expressing the protein, and converting it into a double stranded DNA by a conventional technique.
  • mRNA guanidine isothiocyanate calcium chloride method (Chirwin, et al., Biochemistry, 18, 5294, 1979) and the like can be employed.
  • supports bound with oligo(dT) for example, affinity chromatography in which sepharose or latex particles are used, can be employed.
  • Double stranded cDNA can be obtained by using thus obtained RNA described accordingly as a template to treat with reverse transcriptase, using oligo(dT) that is complementary to poly(A) chain present at 3′-terminus, or a random primer or a synthesized oligonucleotide corresponding to a part of the amino acid sequence of CL-L2s as a primer (Mol. Cell Biol., 2, 161, 1982; Mol. Cell Biol., 3, 280, 1983; Gene, 25, 263, 1983); and by treating thus resulting cDNA strand with for example, E. coli RNaseH, E. coli DNA polymerase 1 , E. coli DNA ligase to alter into the DNA strand.
  • a cDNA library can be produced by incorporating this cDNA into a plasmid vector, a phage vector or a cosmid vector to transform E. coli , or by transfecting it into E. coli following in vitro packaging.
  • the plasmid vector that can be used herein is not particularly limited as long as it can be replicated and maintained in the host.
  • Phage vector is not also particularly limited as long as it can proliferate in the host.
  • Cloning vectors include, for example, pBR322, pUC19, ⁇ gt10, ⁇ gt11 and the like.
  • the vector upon subjecting to immunological screening, has preferably a promoter that enables the expression of a CL-L2s gene in the host.
  • the process for the isolation of cDNA encoding a CL-L2s protein from a cDNA library prepared as described above may involve a general process, which may be used in combination, for the screening of cDNA.
  • a probe labeled with 32 P is produced, and a clone containing the desired cDNA can be screened by a colony hybridization method (Proc. Natl. Acad. Sci. USA, 72, 3961, 1975), or a plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, 2, 108, 1989).
  • a clone may be selected by a PCR method.
  • the desired clone can be selected through the use of an antibody that recognizes CL-L2s when a cDNA library is produced using a vector that can express cDNA.
  • a CL-L2s gene is isolated from cells that express CL-L2s gene, for example, the expressing cells are dissolved using SDS or proteinase K, followed by a phenol treatment. Unwanted RNA is digested with ribonuclease. Thus resultant DNA is digested with restriction enzyme, and the resulting DNA fragments are amplified using phage or cosmid to produce a library. Thereafter, the desired clone is selected, and then a CL-L2s gene can be obtained.
  • the base sequence of the DNA obtained accordingly can be determined by a Maxam-Gilbert method (Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or a Sanger's method (Proc. Nati. Acad. Sci. USA, 74, 5463, 1977).
  • the CL-L2s gene can be obtained by excising from the clone as obtained above.
  • cloning can be also effected by a RT-PCR method using poly(A) + RNA of the cells expressing CL-L2s as a template.
  • the desired cDNA can be also obtained by directly screening the cDNA library after producing/synthesizing a probe based on the base sequence of CL-L2s, not by way of the PCR.
  • the gene of the present invention can be selected among the genes obtained by the methods described herein above through the verification of the base sequence of the gene.
  • the gene of the present invention can be also produced according to the conventional process in which chemical synthesis of a nucleic acid, e.g., phosphoimidite method (Mattencci, M. D. et al., J. Am. Chem. Soc., 130, 3185, 1981) or the like, is employed.
  • phosphoimidite method Moattencci, M. D. et al., J. Am. Chem. Soc., 130, 3185, 1981
  • the present invention also relates to a vector comprising a nucleic acid sequence of CL-L2s.
  • the vector is not particularly limited so far as it can express the CL-L2s protein, however, a plasmid vector, an RNA vector, a DNA vector, a virus vector, a phage vector and the like may be employed.
  • examples thereof include pBAD/His, pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5, pcDNA3.1 or pSecTag2 manufactured by Invirtogen, pET or pBAC manufactured by Novagen Co., pGEM manufactured by Promega, pBluescriptII, pBs, Phagescript, pSG or pSV2CAT manufactured by Stratagene, or pGEX, pUC18/19, pBPV, pSVK3 or pSVL manufactured by Pharmacia Co.
  • the cDNA sequence of CL-L2s ligated to the expression vector is operatively linked to a promoter.
  • the promoter includes for example, phage ⁇ PL promoter, E.coli lac, trp, tac promoter, SV40 early and late promoter, T7 and T3 promoter, retrovirus LTR promoter.
  • the prompter for use in eukaryotic cells include CMV promoter, HSV promoter, SV40 early and late promoter, retrovirus LTR promoter, RSV promoter, metallothionein promoter.
  • the expression vector may contain a marker to allow the selection of the transformed host, and an enhancer.
  • Examples of the marker include dihydrofolate reductase gene, neomycin resistant gene, ampicillin resistant gene and the like.
  • Examples of the enhancer include SV40 enhancer, cytomegalovirus early enhancer promoter, adenovirus enhancer and the like.
  • the present invention further provides transformed cells carrying a base sequence of the present invention to allow the expression thereof by means of the vector as described above that is carrying the base sequence.
  • the host cell for use as a transformed cell in the present invention may preferably include animal cells and insect cells, however, included may be all of the cells (microorganisms may be also included), which can express a CL-L2s protein in the expression vector of the present invention.
  • Exemplary animal cells or insect cells of the present invention may respectively include cells derived from human, or cells derived from fly or silkworm (Bombyx mor).
  • CHO cells, COS cells, BHK cells, Vero cells, myeloma cells, HEK293 cells, HeLa cells, Jurkat cells, mouse L cells, mouse C127 cells, mouse FM3A cells, mouse fibroblast, osteoblast, chondrocyte, S2, Sf9, Sf21, High FiveTM cells may be included.
  • the microorganism according to the present invention include Escherichia coli, Saccharomyces cerevisiae and the like. For the introduction of a vector into such hosts, the method as described above may be employed.
  • the cells expressing CL-L2s of the present invention can be used for analyzing a collectin pathway involving in infectious diseases, immunity and the like. Further, those cells can be utilized in manufacturing a CL-L2s protein or a CL-L2s protein having a carbohydrate chain. The cells can also be utilized in screening for the purpose of obtaining an agonist or an antagonist to the CL-L2s protein.
  • the present invention also relates to a process for the production of a CL-L2s protein which comprises culturing a cell transformed with the base sequence of the present invention as set forth above, and harvesting thus produced CL-L2s.
  • Cell culture, isolation of the protein, and purification thereof may be carried out with conventionally known processes.
  • the protein according to the present invention can be expressed as a recombinant fusion protein, which can be readily isolated, purified, and recognized per se.
  • the recombinant fusion protein is a protein expressed by adding an appropriate peptide chain to the N-terminal end and/or C-terminal end of a protein expressed from a nucleic acid sequence encoding the desired protein.
  • the protein may be expressed as a fusion protein having a signal for extracellular secretion.
  • the protein can be obtained from several kinds of sources such as cultured cells, cultured tissues, transformed cells and the like using conventionally known methods, for example, known purification methods including: salting out such as ammonium sulfate precipitation technique and the like; gel filtration technique using Sephadex and the like; ion exchange chromatographic technique; hydrophobic chromatographic technique; dye gel chromatographic technique; electrophoresis technique; dialysis; ultrafiltration technique; affinity chromatographic technique; high performance liquid chromatographic technique; and the like.
  • known purification methods including: salting out such as ammonium sulfate precipitation technique and the like; gel filtration technique using Sephadex and the like; ion exchange chromatographic technique; hydrophobic chromatographic technique; dye gel chromatographic technique; electrophoresis technique; dialysis; ultrafiltration technique; affinity chromatographic technique; high performance liquid chromatographic technique; and the like.
  • Probes for detecting CL-L2s gene can be specified on the basis of the base sequence set out in either SEQ ID NO: 1, 3, 5, 7, 9, 12, 36, 38 or 40, SEQ ID NO: 48 (corresponding to the base position of 601-1077 of SEQ ID NO: 1) or in the nucleic acid position of 493-969 of SEQ ID NO: 12.
  • primers can be specified for the amplification of DNA or RNA including such a base sequence.
  • An oligonucleotide having a specified base sequence can be obtained through chemical synthesis. When a suitable.
  • the oligonucleotide that is utilized as a primer is of at least 10 bases in length, and suitably of 15 to 50 bases in length. It is desirable that the oligonucleotide that is used as a probe be of from 100 bases to its full length. Further, they can be also used for the diagnosis of diseases caused by mutation of a CL-L2s gene because they can be used for detecting genetic mutation encoding a CL-L2s protein and for detecting SNP. They are expected to be available for the diagnosis of a variety of diseases including for example, bacterial infections and the like. In addition, they are also useful for gene therapy whereby CL-L2s gene is introduced into a living body to allow the expression thereof.
  • promoter region and an enhancer region of the CL-L2s gene that is present in a genome, based on a cDNA base sequence of CL-L2s provided by the present invention.
  • these control regions can be obtained by similar methods to those disclosed in Japanese unexamined patent publication No. 6-181767; J. Immunol., 155, 2477, 1995; Proc. Nati. Acad. Sci, USA., 92, 3561, 1995, and the like.
  • Promoter region referred to herein means a DNA region which controls the expression of a gene that exists upstream of a transcription initiation site.
  • Enhancer region herein refers to a DNA region that enhances the expression of a gene that exists in an intron, a 5′-untranslated region, or a 3′-untranslated region.
  • CL-L2s proteins of the present invention can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and for the development of reagents and drugs for the same. Furthermore, they can be used as an antigen for producing antibodies to CL-L2s. Additionally, they can be utilized in the screening processes of an agonist or an antagonist.
  • the present invention also relates to agonists which stimulate the activity or the activation of CL-L2s of the present invention.
  • the present invention also relates to antagonists which inhibit the activity or the activation of CL-L2s of the present invention.
  • a competitive experimental system can be used, for example, in which mannose or an antibody, and a candidate inhibitor are subjected to the interaction with cells expressing CL-L2s protein thereby allowing the candidate inhibitor to screen based on the binding ratio to mannose. Otherwise, conventionally known methods may also be carried out to effect the screening.
  • the antagonists also include antisense nucleic acids that inhibit the expression of CL-L2s gene. Included in the examples of the other methods for the screening may be methods in which a change in extracellular pH is measured, which is caused by the activation of a receptor (Science, 246, 181-296, 1989), and the like.
  • the present invention relates to transgenic non-human animals having an altered expression level of CL-L2s gene.
  • CL-L2s gene herein includes cDNA, genomic DNA or synthesized DNA encoding hCL-L2s or mCL-L2s.
  • any one of the steps of transcription and translation should be comprised.
  • the transgenic non-human animals according to the present invention are useful for the investigation of functions or expression mechanisms of CL-L2, elucidation of mechanisms of diseases that are anticipated to be involved in CL-L2s, development of diseased animal models for use in screening and safety tests of pharmaceutical products.
  • the gene can be artificially modified to increase or decrease the expression level in comparison with the native expression level of the gene by introducing mutation such as deletion, substitution, addition and/or insertion into a part of some key sites (enhancer, promoter, intron or the like) that regulate the expression of the gene to be proper.
  • mutation such as deletion, substitution, addition and/or insertion into a part of some key sites (enhancer, promoter, intron or the like) that regulate the expression of the gene to be proper.
  • the introduction of the mutation can be carried out by known methods to obtain a transgenic animal.
  • Transgenic animals in their narrow means refer to animals having germ cells into which a foreign gene was artificially introduced by a genetic recombination technique.
  • they include: antisense transgenic animals having a particular gene of which function was suppressed using an antisense RNA; knockout animals having a particular gene knocked out using embryonic stem cells (ES cell); and animals having point mutation of DNA introduced, all of which are animals having a chromosome with a foreign gene being stably introduced therein at an early stage of the development of the individual, and having a genotype that can be transmitted to the progeny thereof.
  • Transgenic animals referred to herein should be comprehended in their broader means including all vertebrates other than human.
  • the transgenic animals according to the present invention are useful for the investigation of functions or expression mechanisms of CL-L2s, elucidation of mechanisms of diseases that are involved in cells expressed in human, development of diseased animal models for use in screening and safety tests of pharmaceutical products.
  • Process for producing a transgenic mouse may include: a process in which a gene is directly introduced into a nucleus of an ovum in a anterior nucleus phase with a micropipette under a phase contrast microscope (microinjection technique, U.S. Pat. No. 4,873,191); a process in which embryonic stem cells (ES cells) are used.
  • ES cells embryonic stem cells
  • a process in which a gene is introduced into a retrovirus vector or an adenovirus vector followed by infection into an ovum; a sperm vector technique in which a gene is introduced into an ovum via a sperm; and the like have been developed.
  • the sperm vector technique is a genetic recombinant process in which a foreign gene is attached to a sperm, or a foreign gene is introduced into a sperm cell with an electroporation technique, and then the foreign gene is introduced into an ovum by fertilizing the ovum (M. Lavitranoet et al., Cell, 57, 717, 1989).
  • site directed genetic recombination in vivo may be also employed by a cre/loxP recombinase system of bacteriophage P1, a FLP recombinase system of Saccharomyces cerevisiae , or the like.
  • a process has been also reported in which a transgene of a desired protein is introduced into a non-human animal using retrovirus.
  • a transgene is required, which is substantially constituted from a promoter involved in expression control, a gene encoding a specified protein, and a poly(A) signal.
  • the manner of the expression and/or the expression level of a specified molecule may be affected by the promoter activity.
  • the manner of the expression and/or the expression level must be confirmed for each of the lineages. Since it has been elucidated that the expression level is altered depending on the untranslated region or splicing, an intron sequence to be spliced at a preceding site of poly (A) signal may be previously introduced.
  • the animal to be used may include mice for use in collecting fertilized ova (5-6 weeks old), male mice for use in mating, female pseudopregnant mice, vas deferens ligated male mice, and the like.
  • gonadotropin or the like may be used for inducing the ovulation.
  • the fertilized ova are harvested, and thereafter, a gene in an injection pipette is introduced into a male pronucleus of the ovum by a microinjection technique.
  • An animal (a pseudopregnant mouse or the like) for use in repositioning the injected ova to an oviduct is provided, to which 10-15 ova are transplanted per one animal.
  • the born mouse can be examined for the introduction of the transgene by: extracting genomic DNA from the end portion of the tail; and detecting the transgene by a Southern method or a PCR technique, alternatively by a positive cloning technique where a marker gene is inserted which is activated upon only the occurrence of homologous recombination.
  • a transcription product derived from the transgene is detected by a Northern method or a RT-PCR technique.
  • a western blotting method may be carried out with a specific antibody to the protein or a fragment thereof.
  • the knockout mouse according to the present invention is one that was treated in a manner to deprive the function of CL-L2s gene.
  • Knockout mouse refers to a transgenic mouse in which an arbitrary gene is destroyed by a homologous recombination technique to impair the corresponding function.
  • the knockout mouse can be produced by homologous recombination using ES cells, followed by the selection of the embryonic stem cell having one of the allelic gene altered/destroyed.
  • a chimeric mouse, which carry cells derived from the embryonic stem cells and cells derived from the embryo being mixed, may be obtained by, for example, injecting the embryonic stem cell that had been genetically engineered at blastocyst stage or morulae stage of the fertilized ovum.
  • this chimeric mouse (chimera refers to a single individual built-up with somatic cells on the basis of more than two fertilized ova) is crossbred with a normal mouse, a heterozygotic mouse can be produced having one of the allelic gene is entirely altered/destroyed. Further, a homozygotic mouse can be produced by crossbreeding heterozygotic mice each other.
  • Homologous recombination refers to the recombination that is caused by a mechanism of genetic recombination between two genes having identical or extremely similar base sequences.
  • PCR can be employed for the selection of cells with the homologous recombination.
  • PCR reaction in which primers corresponding to a part of the inserted gene and a part of the region expected to be inserted are used, may be carried out to reveal the homologous recombination occurring in cells that could yield the amplification products.
  • the gene to be introduced may be joined to a neomycin resistant gene to allow the selection after the introduction into cells by making them resistant to neomycin. Accordingly, known methods and the modified methods thereof can be employed to enable the easy selection.
  • the present invention further provides antibodies that recognize CL-L2s or fragments thereof.
  • the antibodies in accordance with the present invention include for example, the antibodies to a protein comprising an amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13, or a fragment thereof.
  • the antibodies e.g., polyclonal antibodies, monoclonal antibodies, peptide antibodies
  • antisera to CL-L2s or a fragment thereof can be produced using CL-L2s or a fragment thereof of the present invention as an antigen according to any process for producing the antibodies or antisera which is known per se.
  • antibodies that can control the function of CL-L2s e.g., antibodies that recognize CRD, a collagen like domain and the like are useful for pharmaceutical products containing the antibody.
  • CL-L2s or a fragment thereof according to the present invention may be administered neat or with a diluent or a carrier to a warm-blooded animal at a site that enables the production of the antibody upon the administration.
  • complete Freund's adjuvant or incomplete Freund's adjuvant may be administered.
  • the administration may be usually conducted once per 1 to 6 weeks, and two to ten times in total.
  • the warm-blooded animal used may include for example, monkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat, chicken, and the like.
  • mouse and rat may be preferably used.
  • Rat that may be preferably used includes Wistar and SD strain rat
  • mouse that may be preferably used includes BALB/c, C57BL/6 and ICR strain mouse and the like.
  • an individual with the antibody titer that can be recognized therein is selected from the warm-blooded animals e.g., mice that had been immunized with an antigen.
  • spleen or lymph node is collected, and the antibody producing cells contained therein are subjected to the fusion with myeloma cells to effect the preparation of monoclonal antibodies producing cells.
  • the determination of the antibody titer in the antiserum may be carried out for example, by subjecting a labeled CL-L2s described below to a reaction with the antiserum, and thereafter measuring the activity of the label bound to the antibody.
  • the fusion operation can be performed in accordance with a known technique for example, the process of Köhler and Milstein (Nature, 256, 495, 1975) and the modified process thereof (J. Immunol. Method, 39, 285, 1980; Eur. J. Biochem., 118, 437, 1981; Nature, 285, 446, 1980).
  • the fusion accelerating agent may include polyethylene glycol (PEG), Sendai virus and the like, and polyethylene glycol may be preferably used.
  • lectin, poly-L-lysine or DMSO may be added ad libitum to raise the efficiency of the fusion.
  • Examples of the myeloma cell include X-63Ag8, NS-1, P3U1, SP2/0, AP-1 and the like, and SP2/0 may be preferably used.
  • the ratio of antibody producing cell (spleen cell) number to myeloma cell number preferably used is 1:20-20:1.
  • PEG preferably, PEG1000- PEG6000
  • the fusion mixture is incubated at 20-40° C., preferably at 30-37° C. for 1-10 min. Such a condition enables efficient cell fusion.
  • Screening of the hybridoma that produces anti-CL-L2s antibody may be performed by using various methods, which include for example, a process in which a supernatant of hybridoma culture is added to a solid phase (e.g., a microplate) adsorbed with CL-L2s antigen directly or with a carrier, and then an anti-immunoglobulin antibody (when the cells used for the cell fusion was derived from a mouse, anti-mouse immunoglobulin antibody may be used) that was labeled with a radioactive substance, enzyme or the like, or protein A is added thereto thereby detecting the anti-CL-L2s antibody bound to the solid phase; or a process in which a supernatant of hybridoma culture is added to a solid phase adsorbed with an anti-immunoglobulin antibody or protein A, and then CL-L2s labeled with a radioactive substance, enzyme or the like is added thereto thereby detecting the anti-CL-L2s mono
  • Selection and cloning of the anti-CL-L2s antibody can be carried out by known methods per se, or the modified methods thereof. Usually, the process is carried out in a medium for animal cells added with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • the medium for use in the selection, cloning and growing may be any one of the media in which hybridoma can grow.
  • RPMI medium containing 1-20%, preferably 10-20% of fetal bovine serum, GIT medium containing 1-10% of fetal bovine serum, or serum free medium for hybridoma culture, and the like.
  • the temperature of the culture may be preferably about 37° C.
  • the culture period may be usually five days to three weeks, preferably one week to two weeks.
  • the culture is usually conducted in the presence of 5% carbon dioxide gas.
  • the antibody titer of the supernatant of the hybridoma culture can be measured in a similar manner to the measurement of the antibody titer of anti-CL-L2s antibody in an antiserum as described above.
  • a radioimmunoassay (RIA) technique, an enzyme linked immunosorbent assay (ELISA) technique, a FIA (fluorescent immunoassay) technique, a plaque measurement technique, an agglutination reaction technique and the like may be employed as the measurement process, however, the ELISA technique as described below is preferred.
  • the screening by an ELISA technique can be carried out in accordance with the following procedure.
  • a protein which was prepared by a similar process to that for the immunoantigen, is immobilized on the surface of each well of an ELISA plate.
  • BSA, MSA, OVA, KLH, gelatin or skimmed milk or the like is immobilized for the purpose of preventing non-specific adsorption.
  • To each well of this plate added with a supernatant solution of the hybridoma culture, followed by allowing the immunoreaction by standing for a predetermined time.
  • Each well is washed using a washing solution such as PBS or the like.
  • Surfactant may be preferably added to this washing solution.
  • the enzyme for labeling which can be used includes ⁇ -galactosidase, alkaline phosphatase, peroxidase and the like.
  • enzyme reaction is effected through adding a substrate solution of the labeled enzyme that was employed.
  • the enzyme reaction proceeds to change the color of the substrate solution.
  • Cloning can be usually carried out by known methods per se, such as a semisolid agar technique, a limiting dilution technique or the like. Specifically, after the well in which the desired antibody is produced is confirmed by the process described above, a single clone is obtained through conducting the cloning.
  • the process for cloning may involve a limiting dilution technique or the like, in which hybridoma cells are diluted so that one colony per one well of a culture plate is formed, and thereafter the culture is conducted.
  • Cloning by a limiting dilution technique may be performed through the use of feeder cells in order to elevate the colony formation ability, otherwise, a cell growth factor such as interleukin 6 may be added thereto.
  • FACS and single cell manipulation techniques can be employed for the cloning.
  • the cloned hybridoma is cultured preferably in a cell free medium, and an appropriate amount of the antibody is added to the supernatant thereof.
  • resulting single hybridoma may be subjected to a large scale culture using a flask or a cell culture equipment, or may be cultured in the peritoneal cavity of an animal (J. Immunol. Meth., 53, 313, 1982) to give a monoclonal antibody.
  • a medium for cell culture IMDM, DMEM, RPMI 1640, MEM and the like
  • an animal of the same species, and the same strain as the animal from which myeloma cells derived that were used for the cell fusion; otherwise an athymic nude mouse may be preferably used.
  • Hybridoma is transplanted after mineral oil such as pristine or the like is previously administered to the animal.
  • Ascites containing the monoclonal antibody can be obtained after one to two weeks passed, when the myeloma cells enough proliferate.
  • the monoclonal antibody of the present invention can be obtained as the antibody, which does not cross-react with other proteins, by selecting one which recognizes an epitope specific for CL-L2s.
  • an epitope which is presented by serial amino acid residues of at least more that or equal to five, preferably 7 to 20 amino acids among the amino acid sequence constituting the protein, is referred to as an epitope inherent in the protein.
  • the monoclonal antibody that recognizes an epitope constituted from a peptide having an amino acid sequence which were selected from the protein comprising an amino acid set out in any of SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13, and consisting of at least five serial amino acid residues may be identified as the monoclonal antibody specific to hCL-L2s or mCL-L2s according to the present invention.
  • an epitope common to CL-L2s can be selected.
  • a monoclonal antibody can be selected which enables the discrimination of each protein, with a region including an amino acid sequence specific for each of the sequences.
  • the separation and purification of anti-CL-L2s monoclonal antibody can be carried out according to the separation and purification process of an immunoglobulin similarly to the usual separation and purification process of the polyclonal antibodies.
  • Known purification process which can be adopted may include for example, a salt precipitation technique, an alcohol precipitation technique, an isoelectric point precipitation technique, an electrophoretic technique, an ammonium sulfate precipitation technique, an adsorption/desorption technique by an ion exchanger (e.g., DEAE), an ultracentrifugation technique, a gel filtration technique, and a specific purification technique in which an antibody alone is collected by an antigen-bound solid phase or an active adsorbent such as protein A or protein G, or the like, followed by dissociation of the binding to give the antibody.
  • human serum albumin may be added at a concentration of 0.05-2%.
  • amino acids such as glycine, ⁇ -alanine and the like, in particular, basic amino acid such as lysine, arginine, histidine and the like, saccharides such as glucose, mannitol and the like, salts such as sodium chloride may be also added.
  • IgM which is known to be liable to agglutinate, it may be treated with ⁇ -propionolactone and acetic anhydride.
  • the polyclonal antibody according to the present invention can be produced by known methods per se, or the modified methods thereof.
  • an immunoantigen a protein antigen
  • a complex which was formed with the immunoantigen and a carrier protein
  • the antibody is purified/isolated.
  • the kind of the carrier protein and the mixing ratio of the carrier and hapten may be optionally determined as long as the antibody can be efficiently produced to the hapten subjected to the immunization after crosslinking with the carrier.
  • any kind of the carrier protein may be crosslinked at any ratio, however, the process in which about 0.1-20, preferably about 1-5 of bovine serum albumin, bovine thyroglobulin, hemocyanin or the like, for example, is coupled with 1 of hapten by weight may be used.
  • various condensing agents may be used for the coupling of the hapten and carrier, which may include glutaraldehyde and carbodiimide, and active ester reagents containing maleimide active ester, thiol group, dithiopyridyl group and the like.
  • the condensation product is administered neat or with a carrier or a diluent to a warm-blooded animal at a site that enables the production of the antibody upon the administration.
  • complete Freund's adjuvant or incomplete Freund's adjuvant may be administered.
  • the administration may be usually conducted once per 2 to 6 weeks, and three to ten times in total.
  • Polyclonal antibodies can be collected from the blood, ascites and the like, and preferably from the blood, of the warm-blooded animal immunized by a process as described above.
  • the measurement of the antibody titer in antiserum can be carried out in a similar manner to the measurement of the antibody titer of the antiserum as described above.
  • the separation and purification of the polyclonal antibody can be carried out according to the separation and purification process of an immunoglobulin similarly to the separation and purification process of a monoclonal antibody described above.
  • Monoclonal antibodies and polyclonal antibodies to CL-L2s or a fragment thereof can be utilized in diagnosis and therapy of the diseases relating to the cells that are expressing CL-L2s.
  • CL-L2s or a fragment thereof can be measured using these antibodies, on the basis of the immunological binding with CL-L2s or the fragment thereof according to the present invention.
  • the process for measuring CL-L2s or a fragment thereof using such an antibody may include for example, sandwich techniques in which a sandwich complex is detected which was produced by subjecting CL-L2s or a fragment thereof to a reaction with an antibody coupled to an insoluble support and a labeled antibody; or competitive techniques in which CL-L2s or a fragment thereof in a sample is measured by subjecting labeled CL-L2s, and CL-L2s or a fragment thereof in a sample to a competitive reaction with the antibody followed by the measurement of CL-L2s or a fragment thereof in a sample from the amount of the labeled antigen that reacted with the antibody.
  • Insoluble support for use in the measurement include for example, synthetic resin such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylic acid ester, nylon, polyacetal, fluorine-contained resin and the like; polysaccharides such as cellulose, agarose and the like; glasses; metals; and the like.
  • the insoluble support may be in a variety of forms, and for example, tray-like, spherical, fibrous, cylindrical, discal, vessel-like, cell-like, tubular, and the like may be adopted.
  • the support onto which the antibody had been adsorbed may be stored ad libitum in cold, in the presence of an antiseptic agent such as sodium azide and the like.
  • Chemical coupling process includes for example, processes in which glutaraldehyde is used; maleimide processes in which N-succinimidyl-4-(N-maleimidemethyl) cyclohexane-1-carboxylate and N-succinimidyl-2-maleimide acetate and the like are used; carbodiimide processes in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and the like is used.
  • process includes maleimidebenzoyl-N-hydroxysuccinimide ester processes, N-succimidyl-3-(2-pyridylthio) propionic acid processes, bisdiazobenzidine processes, dipalmityl lysine processes.
  • a complex that had been formed previously by subjecting the substance to be detected to a reaction with two kinds of antibodies of which epitopes are different can be captured by the third antibody to the antibody, which had been immobilized in a similar manner to those described above.
  • the material to be used for labeling may include enzyme, fluorescent materials, luminescence materials, radioactive materials, metal chelates and the like.
  • enzyme may include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, ⁇ -glycerolphosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, asparaginase, glucose oxidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase and the like.
  • Fluorescent materials may include for example, fluorescein isothiocyanate, phycobilin protein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalic aldehyde and the like.
  • Luminescence materials may include isoluminol, lucigenin, luminol, aromatic acridinium esters, imidazole, acridinium salts and modified esters thereof, luciferin, luciferase, aequorin and the like.
  • Radioactive materials may include 125 I, 127 I, 131 I, 14 C, 3 H, 32 P, 35 S and the like. These materials are not limited thereto as long as the material can be used in immunological determination methods.
  • low molecular weight hapten such as biotin, dinitrophenyl, pyridoxal or fluorescamine may be conjugated to the antibody.
  • horseradish peroxidase may be used as a labeling enzyme. This enzyme can react with many kinds of substrates, which can be readily conjugated to the antibody by a periodic acid method.
  • an enzyme When an enzyme is used as a labeling agent, a substrate for measuring its activity, and a color-developing agent as needed may be employed.
  • a substrate for measuring its activity When peroxidase is used as an enzyme, H 2 O 2 may be used as a substrate solution, and 2,2′-azino-di-[3-ethylbenzthiazolin sulfonate] ammonium (ABTS), 5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine, 3,3′,5,5′-tetramethylbenzidine or the like may be used as a color-developing agent.
  • ABTS 2,2′-azino-di-[3-ethylbenzthiazolin sulfonate] ammonium
  • kit products including a monoclonal antibody, a polyclonal body described above, and reagents.
  • available crosslinking agents include N,N′-orthophenylenedimaleimide, 4-(N-maleimidemethyl) cyclohexanoyl N-succinimide ester, 6-maleimidehexanoyl N-succinimide ester, 4,4′-dithiopyridine, and other known crosslinking agents.
  • the reaction of such a crosslinking agent with the enzyme and the antibody may be conducted in accordance with known methods depending upon the properties of the respective crosslinking agents.
  • the antibodies to be used may be any fragments of the antibodies for example, Fab′, Fab, F(ab′) 2 depending on the condition.
  • enzymatically labeled antibodies may be prepared by using a similar process to any one of those for polyclonal antibodies and monoclonal antibodies.
  • the enzymatically labeled antibody that was obtained by using the aforementioned crosslinking agent is purified by any known methods such as affinity chromatography or the like, more sensitive immunological determination system can be achieved.
  • the enzymatically labeled antibody, which was purified in such a manner is stored in a cold and dark place after adding thimerosal, glycerol or the like as a stabilizer, alternatively, after being lyophilized.
  • the subject sample for the measurement may be a sample containing CL-L2s, which may include body fluids such as plasma, serum, blood, urine, tissue fluid, cerebrospinal fluid and the like, various types of cells, tissues, and the like.
  • body fluids such as plasma, serum, blood, urine, tissue fluid, cerebrospinal fluid and the like, various types of cells, tissues, and the like.
  • Process for the production of human monoclonal antibodies involves transformation techniques with Epstein-Barr virus (EBV), and fusion techniques in which thus transformed cells and parent cells are fused; processes in which a chimeric antibody or a humanized antibody is produced using genetic engineering techniques; and the like in addition to cell fusion techniques.
  • Chimeric antibody refers to an antibody that was produced by linking immunoglobulin gene fragments from heterogeneous animals.
  • Humanized antibody refers to an antibody having a substituted primary structure in part other than a complementarity determining region (CDR) of H chain and L chain with the corresponding primary structure of a human antibody through introducing the alteration to a mouse antibody or the like that is heterogeneous to human.
  • CDR complementarity determining region
  • a mouse is immunized first, and an antibody variable region (V region) that binds to an antigen is excised from a gene of the mouse monoclonal antibody. Thereafter, the V region is linked to a gene of an antibody constant region (C region) derived from human myeloma to give a chimeric gene.
  • V region antibody variable region
  • C region antibody constant region
  • human-mouse monoclonal antibody can be produced. Because chimeric antibodies are less antigenic to human, they can be utilized as a monoclonal antibody for therapeutic use to be administered into a human body, or for use in diagnostic imaging.
  • Known techniques relevant to chimeric antibodies involve Japanese patent unexamined publication No.
  • Humanized antibodies were recently developed, which are appreciated as being more useful than chimeric antibodies.
  • Humanized antibody refers to an antibody that is humanized as a whole molecule except for CDR of an antibody molecule by grafting only a sequence of a gene for an antigen-binding site (CDR: complementarity determining region) of an antibody molecule into a gene of a human antibody (CDR grafting). This antibody is appreciated as being safer with less antigenicity than the human-mouse chimeric antibody because it has less part derived from a mouse antibody.
  • SHM-D 33 strain ATCC CRL 1668
  • RF-S1 strain both of which being human/mouse heteromyeloma
  • high fusion efficiency can be achieved that is equivalent to mouse parent cells.
  • Hybridoma that was obtained using these parent cells can be cloned without feeder cells, and it can produce IgG type antibody in a comparatively stable manner at a large amount.
  • ERDF medium supplemented with 15% FCS may be used, although other operation may be similarly carried out to the operation for the murine cells.
  • human lymphocytes collected from peripheral blood may be preferably employed, which were sufficiently sensitized with an antigen.
  • sensitization with an antigen may be also conducted in vitro. In Japan, clinical trials have been currently carried out for humanized antibodies to adult T cell leukemia.
  • the antibodies according to the present invention can be humanized, and such antibodies would be extremely useful upon the administration to human.
  • CL-L2s polynucleotides or proteins are possibly utilized in diagnostic, prophylactic and therapeutic methods, and for the development of reagents and drugs for various types of diseases including bacterial infections and the like.
  • composition according to the present invention may comprise CL-L2s polynucleotides or proteins, substances that stimulate or inhibit the activity or activation of CL-L2s protein, substances including antibodies to CL-L2s protein and the like (hereinafter, referred to as “CL-L2s related substance”).
  • CL-L2s related substances can be used neat, or after subjecting to several kinds of treatment such as dilution in water and the like, however, they can be used after blending in pharmaceutical products, quasi drugs and the like. In these cases, the amount of the substance to be blended may be determined ad libitum. When the substance is formulated for the systemic administration, 0.001-50% by weight, in particular, 0.01-10% by weight is permissible. When the amount is less than 0.001%, sufficient action of lacrimation may not be enabled. When the amount is greater than 50%, properties such as stability, flavor and the like of the composition itself may be deteriorated.
  • the route of administration can be optionally selected from the administration via mucosa, transdermal administration, intramuscular administration, subcutaneous administration, endorectal administration, topical ocular administration, and the like, in addition to oral administration and intravenous administration described above.
  • the CL-L2s related substance according to the present invention may be included in the formulation as a salt.
  • Pharmaceutically acceptable salts include for example, salts with base such as inorganic base, organic base and the like; acid addition salts such as those of inorganic acid, organic acid, basic or acidic amino acid.
  • Inorganic bases include for example, alkaline metal such as sodium, potassium and the like; alkaline earth metal such as calcium, magnesium and the like; aluminum, ammonium and the like.
  • Organic bases include for example, primary amines such as ethanolamine and the like; secondary amines such as diethylamine, diethanolamine, dicyclohexylamine, N,N′-dibenzylethylenediamine and the like; tertiary amines such as trimethylamine, triethylamine, pyridine, picoline, triethanolamine and the like.
  • Inorganic acids include for example, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • Organic acids include for example, formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, benzoic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like.
  • Basic amino acids include for example, arginine, lysine, ornithine and the like.
  • Acidic amino acids include for example, aspartic acid, glutamic acid and the like.
  • Examples of dosage forms for use in oral administration include powdered formulations, granulated formulations, encapsulated formulations, pills, tablets, elixirs, suspensions, emulsions, syrups and the like, which may be selected ad libitum.
  • such formulations may be modified, which may involve release control, stabilization, facilitation of disintegration, blocking of disintegration, enteric coating, facilitation of absorption and the like.
  • examples of dosage forms for the intraoral topical administration include chewable formulations, sublingual formulations, buccal formulations, lozenges, ointments, plasters, liquid formulations and the like, which may be selected ad libitum.
  • such formulations may be modified, which may involve release control, stabilization, facilitation of disintegration, blocking of disintegration, enteric coating, facilitation of absorption and the like.
  • DDS formulation referred to herein involves sustained release formulations, topically applied formulations (lozenges, buccal formulations, sublingual formulations), drug controlled release formulations, enteric coated formulations, formulations soluble in stomach and the like, which are formulations that are prepared so that most appropriate dosage form is accomplished taking into account of the administration route, bioavailability, adverse effect and the like.
  • Components for DDS essentially comprise a drug, a drug release module, a coating and a therapy program.
  • the drug having a short half life is preferred, which permits rapid decline of the blood concentration particularly upon cessation of the release thereof.
  • the coating is preferably nonreactive to the body tissue of the part to which the drug is administered.
  • the therapy program is preferably configured so that the most optimal drug concentration is kept during the predetermined period.
  • the drug release module substantially has a drug storage, a release control part, an energy source, and a release opening or a release surface. All of these fundamental components are not necessarily required, and thus addition, deletion or the like may be optionally carried out to select the best mode.
  • Examples of materials which can be used for DDS include polymers, cyclodextrin derivatives, lecithin and the like.
  • the polymer may include insoluble polymers (silicone, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylcellulose, cellulose acetate and the like), water soluble polymers and hydroxyl gel-forming polymers (polyacrylamide, polyhydroxyethyl methacrylate cross-linked form, polyacryl cross-linked form, polyvinyl alcohol, polyethyleneoxide, water soluble cellulose derivatives, cross-linked poloxamer, chitin, chitosan and the like), slow dissolving polymers (ethyl cellulose, a partial ester of methylvinyl ether-maleic anhydride copolymer and the like), polymers soluble in stomach (hydroxylpropylmethyl cellulose, hydroxylpropyl cellulose, carmellose sodium, macrogol, polyvinylpyrrol
  • silicone, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, a partial ester of methylvinyl ether-maleic anhydride copolymer can be used for the control of drug release;
  • cellulose acetate can be used as a material of a osmotic pressure pump;
  • ethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose can be used as a material of a membrane of slow dissolving formulations;
  • polyacryl cross-linked form can be used as an attaching agent to mucosa.
  • the formulation can be manufactured with adding solvent, excipient, coating agent, base, binding agent, lubricant, disintegrant, solubilizing agent, suspending agent, thickening agent, emulsifying agent, stabilizing agent, buffering agent, isotonizing agent, soothing agent, preservative agent, flavoring agent, fragrance agent, coloring agent and the like in compliance with its dosage form (known dosages form such as forms for oral administration, injection, suppository and the like).
  • dosage form such as forms for oral administration, injection, suppository and the like.
  • [0170] purified water, water for injection, saline, peanut oil, ethanol, glycerol;
  • [0171] starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose, xylitol;
  • [0173] vaseline, vegetable oil, macrogol, base for oil in water emulsion, base for water in oil emulsion;
  • [0174] [binding agent] natural polymer compounds such as starch and derivatives thereof, cellulose and derivatives thereof, gelatin, sodium alginate, gum tragacanth, gum arabic, and the like; synthetic polymers such as polyvinylpyrrolidone and the like; dextrin, hydroxylpropyl starch;
  • [0177] [solubilizing agent] cyclodextrin, ethanol, propylene glycol, polyethylene glycol;
  • [0178] gum arabic, gum tragacanth, sodium alginate, aluminum monostearate, citric acid, various surfactants;
  • [0180] emulsifying agent] gum arabic, cholesterol, gum tragacanth, methyl cellulose, various surfactants, lecithin;
  • [0183] [isotonizing agent] sodium chloride, glucose;
  • [0185] benzoic acid and salts thereof, p-hydroxybenzoic esters, chlorobutanol, inverted soap, benzyl alcohol, phenol, thimerosal;
  • [0188] water soluble edible dye, lake dye.
  • Novel collectin according to the present invention is described in more detail by the following non-limiting illustrative examples. However, the present invention should not be construed to be limited by the examples.
  • Example 1 search on EST database (Example 1); screening of a novel human collectin from a cDNA library derived from human liver by PCR and sequencing of the base sequence (Example 2); screening of a novel human collectin a cap site cDNA library derived from human kidney by PCR and sequencing of the base sequence (Example 3); homology search of the novel collectin (Example 4); obtaining the novel mouse collectin cDNA (Example 5); analysis of distribution of expression of the novel human collectin in human tissues (Example 6); genetic analysis of the novel human collectin (Example 7); analysis of distribution of expression of the novel collectin CL-L2-1 and CL-L2-2 in human tissues (Example 8); construction of an expression vector pcDNA3.1/Myc-His(+)-CL-L2-1,2 of the novel collectin (Example 9); production of a cell strain that is stably expressing the novel collectin (Example 10); analysis of sugar specificity of the novel collectin (Example 1); screening of
  • FIGS. 2 and 3 illustrate homology of amino acid residues of known collecting, i.e., human MBP, human SP-A, human SP-D and collectin CL-L1 derived from human liver, which was successfully isolated by the present inventor recently (see, Japanese patent unexamined publication No. Hei 11-206377), having a common structure as depicted in FIG. 1.
  • portions of amino acid residues that are recognized as being homologous were boxed.
  • An amino acid sequence of CRD which is responsible for a lectin activity (SEQ ID NO: 14) of CL-L1 in this Figure was used to search on EST (Expressed Sequence Tags) database.
  • a consensus sequence (SEQ ID NO: 15) was produced in view of base sequences of 10 clones described above. Then, two primers toward the upstream direction: CAP1 (5′-agattttattgtatagcttgg-3′ (SEQ ID NO: 16)) and CAP2 (5′-ctgggtaataattacataatg-3′ (SEQ ID NO: 17) on the basis of a consensus sequence obtained in Example 1, and primes pertinent to a part of a region of a vector of the cDNA library derived from human liver: ⁇ TriplEx-F1 (5′-aagctccgagatctggacgag-3′ (SEQ ID NO: 18)) and ⁇ TriplEx-F2 (5′-ctcgggaagcgcgccattgtg-3′ (SEQ ID NO: 19)) were synthesized with 392A DNA/RNA synthesizer manufactured by PE Applied Biosystems Inc., and the
  • First PCR was conducted using a cDNA library derived from human (Clontech Co.,) as a template of the screening by PCR.
  • Reaction mixture contained LA PCR Buffer II (Mg 2+ free), 2.5 mM MgCl 2 , each 1 ⁇ L of 200 ⁇ M dATP, dCTP, dGTP and dTTP (any of which is manufactured by Takara Shuzo Co., Ltd.), a cDNA library derived from human liver (Clontech Co.,), 0.5 ⁇ M ⁇ TriplEx-F1 primer and 0.5 ⁇ M CAP1 primer in a total volume of 50 ⁇ L.
  • PCR was performed with a program of 35 cycles of heat denaturation at 95° C.
  • first PCR second PCR was conducted.
  • the product of the first PCR was used as a template at 1 ⁇ L, and primers employed were ⁇ TriplEx-F2 primer and CAP2 primer.
  • the reaction was performed with a similar reaction constitution and program to the first PCR except that the cycle number was 25 cycles.
  • the PCR hereinabove was performed with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc.
  • PCR product was verified on an agarose gel electrophoresis, and excised from the gel followed by freezing at ⁇ 80° C. for 10 min. After centrifuging at 15000 rpm for 10 min, the product was purified by ethanol precipitation of the supernatant.
  • the purified DNA fragment was incorporated into pT7Blue Vector manufactured by Novagen CO., and the vector was transformed into competent cells, XL1-Blue cells.
  • the transformant was cultured in a LB medium (100 ⁇ g/mL ampicillin) followed by extraction of the plasmid with an alkaline SDS method to sequence the base sequence thereof with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by Applied Biosystems Inc.
  • Reaction mixture contained LA PCR Buffer II (Mg 2+ free), 2.5 mM MgCl 2 , each 1 ⁇ L of 200 ⁇ M dATP, dCTP, dGTP and dTTP (any of which is manufactured by Takara Shuzo Co., Ltd.), a Cap Site cDNA Human kidney, 0.5 ⁇ M 1RC2 primer (both of which were manufactured by NIPPON GENE Co., Ltd.), and 0.5 ⁇ M CAP3 primer in a total volume of 50 ⁇ L.
  • PCR was performed with a program of 35 cycles of heat denaturation at 95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation reaction at 72° C.
  • first PCR second PCR was conducted.
  • the product of the first PCR was used as a template at 1 ⁇ L, and primers employed were attached 2RC2 primer (5′-gtacgccacagcgtatgatgc-3′ (SEQ ID NO: 25)) and CAP4 primer.
  • the reaction was performed with a similar reaction constitution and program to the first PCR except that the cycle number was 25 cycles.
  • the PCR hereinabove was performed with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc.
  • PCR product was verified on an agarose gel electrophoresis, and excised from the gel followed by freezing at ⁇ 80° C. for 10 min. After centrifuging at 15000 rpm for 10 min, the product was purified by ethanol precipitation of the supernatant.
  • the purified DNA fragment was incorporated into pT7Blue Vector manufactured by Novagen CO., and the vector was transformed into competent cells, XLI-Blue cells.
  • the transformant was cultured in a LB medium (100 ⁇ g/mL ampicillin) followed by extraction of the plasmid with an alkaline SDS method to sequence the base sequence thereof with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by Applied Biosystems Inc.
  • Primers employed were M13 Universal Primer (5′-cgacgttgtaaaacgacggccagt-3′ (SEQ ID NO: 20)) and M13 Reverse Primer (5′-caggaaacagctatgac-3′ (SEQ ID NO: 21)).
  • Two base sequences accordingly obtained were revealed to be: a base sequence having 492 bases longer than the base sequence obtained in Example 2 to its N-terminal direction (SEQ ID NO: 1); and a base sequence having 274 bases longer than the base sequence obtained in Example 2 to its N-terminal direction (SEQ ID NO: 3).
  • mCL-L2 gene could be obtained through the screening of a mouse liver cDNA library.
  • the resulting cDNA clone of mCL-L2 was confirmed to have an ORF (open reading frame) of 813 bases (SEQ ID NO: 12), and encode amino acids of 271 amino acids set out in SEQ ID NO: 13.
  • RT-PCR was performed using two primers which are capable of amplifying a cDNA sequence spanning from neck region to carbohydrate recognition domain of the novel collectin: RTF1 (5′-agattccggtgacataggacc-3′ (SEQ ID NO: 26)), RTR1 (5′-tggtctgggctctgtccctgc-3′ (SEQ ID NO: 27)), and two primers which are capable of amplifying a part of ⁇ -actin gene for use in comparison of the amount of expressed novel collectin in each of the tissues: human ⁇ -actin sense primer (5′-caagagatggccacggctgct-3′ (SEQ ID NO: 28)), human ⁇ -actin antisense primer (5′′-tccttctgcatcctgtcggca-3′ (SEQ ID NO: 29)). All of these primers were synthed.
  • RT-PCR was carried out using RNA LA PCR Kit (AMV) Ver.1.1 (TAKARA Syuzo, Co.) with each RNA derived from several human tissues ((1) brain, (2) heart, (3) kidney, (4) liver, (5) lung, (6) trachea, (7) bone marrow, (8) colon, (9) small intestine, (10) spleen, (11) stomach, (12) thymus, (13) mammary gland, (14) prostate gland, (15) skeletal muscle, (16) testis, (17) uterus, (18) cerebellum, (19) fetal brain, (20) fetal liver, (21) spinal cord, (22) placenta, (23) adrenal gland, (24) pancreas, (25) salivary gland, and (26) thyroid) as a template.
  • AMV RNA LA PCR Kit
  • Ver.1.1 TAKARA Syuzo, Co.
  • the reaction mixture contained 5 mM MgCl 2 , 1 ⁇ RNA PCR Buffer, 1 mM dNTP Mixture, 1 U/ ⁇ l RNase inhibitor and 2 ⁇ g of RNA, and total volume of the mixture was adjusted to give 40 ⁇ l with RNase free distilled water.
  • a reaction mixture without reverse transcriptase was also prepared for a negative control.
  • the reaction mixture as described above was placed in 0.2 ml tube, and subjected to a reverse transcription reaction with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc. through 1 cycle of: 30 minutes at 42° C., 5 minutes at 99° C., and 5 minutes at 5° C.
  • resulting reverse transcription reaction product was subsequently used at 10 ⁇ L for LA PCR in the following reaction mixture with respectively 28 cycles. and 35 cycles. Thereto were added 2.5 mM MgCl 2 , 1 ⁇ LA PCR Buffer (Mg 2+ free), 2U TaKaRa LA Taq, 0.2 ⁇ M RTF1 primer and 0.2 ⁇ M RTR1 primer, and the mixture was adjusted to give total volume of 50 ⁇ L with sterilized distilled water.
  • PCR was performed with a program of 28 or 35 cycles of heat denaturation at 95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation reaction at 72° C. for 60 seconds; and in addition, heat denaturation at 95° C.
  • RT-PCR was performed to amplify a part of ⁇ -actin with each of the tissues, and the correction of the amount of RNA was conducted.
  • the RT-PCR was performed similarly to the above procedure with reverse transcription reaction, PCR, and the estimation was executed. The results are illustrated in FIG.
  • Collectins targeted for the analysis were proteins in various collecitn families illustrated in FIG. 8 (in the Figure, CL-L1 and CL-P1 were successfully isolated by the present inventors). Multiple alignment was produced with clustalw method using a region containing a lectin domain on the basis of data which were obtained by searching each amino acid sequences from GenBank database. A phylogenetic tree was produced with Phylip version 3.57 c package program using N-J process (neighbor-joining process) based on thus resulting multiple alignment.
  • RT PCR was conducted using primers which are capable of amplifying the entire translated region of CL-L2-1 as obtained (RTF2 (5′-atgagggggaatctggccctggtg-3′ (SEQ ID NO: 30)), RTR2 (5′-catgttctccttgtcaaactcac-3′ (SEQ ID NO: 31))); primers which are capable of amplifying the entire translated region of CL-L2-2 as obtained (RTF3 (5′-atgtggtgggtgcctccgagtc-3′ (SEQ ID NO: 32)), RTR2 (SEQ ID NO: 31)); and two human ⁇ -actin primers used in Example 6, which are capable of amplifying a part of ⁇ -actin gene for use in comparison of the amount of expressed novel
  • FIG. 9 The results are illustrated in FIG. 9, which demonstrate intensive expression of CL-L2-1 (SEQ ID NO: 1) according to the present invention for PCR performed with 28 cycles in kidney (lane 3), liver (lane 4), small intestine (lane 9), thymus (lane 12), fetal liver (lane 20), spinal cord (lane 21), adrenal gland (lane 23), pancreas (lane 24). Further, with respect to PCR performed with 35 cycles, ubiquitous expression of CL-L2-1 (SEQ ID NO: 1) could be verified in all tissues tested, although varying intensity of expression was observed.
  • CL-L2-2 (SEQ ID NO: 3) was intensively expressed in kidney (lane 3) for PCR performed with 28 cycles, whilst for PCR performed with 35 cycles was expressed in kidney (lane 3) as well as in prostate gland (lane 14), testis (lane 16), spinal cord (lane 21), placenta (lane 22).
  • Base sequence was determined with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by PE Applied Biosystems Inc. Primers employed were M13 Universal Primer (5′-cgacgttgtaaaacgacggccagt-3′ (SEQ ID NO: 20)) and M13 Reverse Primer (5′-caggaaacagctatgac-3′ (SEQ ID NO: 21)).
  • CL-L2-1v1 has deletion of the amino acid position of 44-91 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 394-537 of CL-L2-1 set out in SEQ ID NO:1), whose amino acid sequence is encoded by the position of 265-933 of a base sequence set out in SEQ ID NO: 36 (SEQ ID NO: 59).
  • CL-L2-1v2 has deletion of the amino acid position of 44-67 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 394-465 of CL-L2-1 set out in SEQ ID NO:1), whose amino acid sequence is encoded by the position of 265-1005 of a base sequence set out in SEQ ID NO: 38 (SEQ ID NO: 60).
  • CL-L2-1v3 has deletion of the amino acid position of 68-91 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 466-537 of CL-L2-1 set out in SEQ ID NO: 1), whose amino acid sequence is encoded by the position of 265-1005 of a base sequence set out in SEQ ID NO: 40 (SEQ ID NO: 61).
  • mCL-L2-2 gene can be obtained in a similar process to that in Example 4.
  • a translated region of CL-L2-1 (SEQ ID NO: 1) was amplified using CL-L2-1F primer (5′-gggaagcttcgatcaggatgagggggaatctggcctggtg-3′ (SEQ ID NO: 33)) and CL-L2-1R primer (5′-gggctcgagcatgttctccttgtcaaactcac-3′ (SEQ ID NO: 34)) by PCR (Takara Thermal Cycler MP manufactured by Takara Shuzo Co., Ltd.) with a cDNA library derived from human kidney as a template.
  • a translated region of the novel collectin (SEQ ID NO: 3) was amplified using CL-L2-2F primer (5′-gggaagcttccagcacaatgtggtgggtgcctccgagtc-3′ (SEQ ID NO: 35)) and CL-L2-1R primer (SEQ ID NO: 34) by PCR with a cDNA library derived from human kidney as a template.
  • CL-L2-1 cDNA was ligated to pT7Blue T-Vector (manufactured by Novagen Co.) and was transformed into Escherichia coli XLI-Blue.
  • a plasmid containing CL-L2-1 cDNA was purified from the resulting clone.
  • the plasmid with no error was digested with restriction enzymes Hind III and Xho I, and ligated to pcDNA3.1/Myc-His(+)A vector (manufactured by Invitrogen Co,.) that had been digested with the same enzymes and purified.
  • the ligated plasmid was transformed into Escherichia coli , XLI-Blue, the resulting clone was cultured. The plasmid was then purified to give an expression vector pcDNA3.1/Myc-His(+)A-CL-L2-1,2.
  • expression vectors were similarly produced for variants of CL-L2-1 and CL-L2-2 (SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40).
  • Transient expression was conducted through cotransfection of the expression vector pcDNA3.1/Myc-His(+)A-CL-L2-1 obtained in Example 9 and pEGFP-F vector (manufactured by Clontech Co.,) to CHO cells using LIPOFECTAMINE 2000 (LF2000) Reagent (manufactured by GIBCO BRL Co.).
  • LIPOFECTAMINE 2000 (LF2000) Reagent manufactured by GIBCO BRL Co.
  • a 0.5 ml solution of LF2000 Reagent (LF2000 Reagent 30 ⁇ l, Nutrient Mixture F-12 Ham (Ham's F-12 medium, (manufactured by Sigma Co.)) was first prepared, and incubated at room temperature for 5 minutes.
  • a vector solution (pcDNA3.1/Myc-His(+)A-CL-L2-1,2: 7.5 ⁇ g, pEGFP-F vector 2.5 ⁇ g, Ham's F-12 medium) was admixed therewith, followed by incubation for 20 minutes. Thereafter, the solution was added to CHO cells that had been cultured to a high density in a 25 cm 2 flask including 5 ml of Ham's F-12 medium containing 5% FCS. After incubating at 37° C. for 4 hours in the presence of 5% CO 2 , the medium was replaced with a flesh medium, followed by subsequent incubation at 37° C. for 20 hours in the presence of 5% CO 2 .
  • the medium was replaced with Ham's F-12 medium containing 5% FCS, 0.4 mg/ml Geneticin, (manufactured by GIBCO BRL Co.), and 10 days culture was subsequently conducted. In this process, replacement of the medium was once carried out.
  • each 100 ⁇ l of a culture medium was further added thereto followed by the additional culture for one week.
  • a clone proliferated by the drug selection with Geneticin was divided into two parts, which were subjected to passages on 12-well and 24-well cell culture plates. Upon the passage, clones were excluded, which were derived from proliferation in a well where two or more cells existed per well, and the cells were plated at a cell number ratio of 9:1 for the 12-well and 24-well cell culture plates. The cells were cultured at 37° C. in the presence of 5% CO 2 until the cells in the 12-well plate reach to high density.
  • the membrane was washed with 100 ml of 0.05% Tween 20/TBS buffer at room temperature for 20 min. ⁇ 3 followed by further incubation in a solution of anti-IgG-HRP (manufactured by Chemicon Co., Ltd.) ⁇ 5000 diluted in 0.05% Tween 20/TBS buffer at room temperature for 1 hour. Thereafter, the membrane was washed with 100 ml of 0.05% Tween 20/TBS buffer at room temperature for 20 minutes ⁇ 3 followed by detection using TMB Membrane Peroxidase substrate system (manufactured by Funakoshi KK). After confirming the clones with intense color development, cells in respectively corresponding wells of the 24-well plate were identified as a stably expressing cell strain (CHO/CL-L2-1).
  • Ni-NTA agarose was packed in Poly-Prep Chromatography Columns (manufactured by BIO-RAD Co., Ltd.) followed by washes with 5 ml of 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, 0.05% Tween20 (pH8.0) three times, and by elution with 200 ⁇ l of 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, 0.05% Tween20 (pH8.0) five times to purify the novel collectin. Purified novel collectin was quantitatively determined, and used for the analysis of sugar specificity.
  • the membrane was washed with 0.05% Tween20, 5 mM CaCl 2 , TBS buffer three times, and was incubated in each sugar chain probe solution (see, FIG. 10) diluted with 5 mM CaCl 2 , TBS buffer to give 1 ⁇ g/ml at room temperature for 1 hour. Thereafter, the membrane was again washed with 0.05% Tween20, 5mM CaCl 2 , TBS buffer three times. Then the membrane was incubated in a solution of Streptabvidin-biotinylated HRP (manufactured by Amersham Co.) ⁇ 1000 diluted in 0.05% Tween20, 5 mM CaCl 2 , TBS buffer at room temperature for 30 min.
  • Streptabvidin-biotinylated HRP manufactured by Amersham Co.
  • the novel collectin was active in binding to mannose, fucose, N-acetylgalactosamine, N-acetylneuraminic acid, mannose-6-phosphoric acid.
  • CL-L2s protein of the present invention has a collectin structure, it is believed to be a substance that exerts characteristic effects to those structures. Therefore, it can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and in the development of reagents and drugs for the same.

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Abstract

Provided are isolated collectin (CL-L2s) genes including a base sequence set out in SEQ ID NO: 1, 3, 5, 7, 9, 12, 36, 38 or 40 relating to a novel collectin which are expected to exhibit an antibacterial activity, an antiviral activity and the like particularly in a human body; and isolated collectin proteins including an amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41 and derivatives and fragments thereof.

Description

    FIELD OF THE INVENTION
  • The present invention relates to isolated human and mouse collectins (herein referred to as “hCL-L2” and “mCL-L2” respectively, or merely as “CL-L2” when discrimination of both is not intended), genes and proteins, the homologues, mutants, modified forms and polymorphic variants thereof (these are collectively referred to as “derivatives”), fragments thereof (hereinafter collectively referred to as “CL-L2s” for all of these), and the detection of the same. The present invention further relates to compositions which comprise CL-L2s for pharmaceutical use, diagnostic use and research use, and methods for the production and use of the same. Additionally, the present invention relates to agonists and antagonists of CL-L2s proteins, as well as methods for screening drugs using CL-L2s. Moreover, the present invention also relates to expression vectors comprising CL-L2s gene, transformed cells that were transformed with the expression vector, antibodies to CL-L2s protein, and cells that produce the antibody. [0001]
    • BACK GROUND OF THE INVENTION
  • Complement systems that play an important role in a body defense mechanism are known to include: a classic pathway in which an immunoglobulin serves as a recognition molecule followed by the activation of C1 that is the first component of the complement; and an alternative pathway in which C3, which is the third component of the complement, is directly coupled to foreign substances such as bacteria. In addition to these pathways of the complement activation, a lectin pathway was illustrated in which a mannose binding protein (hereinafter referred to as “MBP”), which is a serum lectin, activates the complement system through the direct recognition of and coupling with a carbohydrate chain on the surface of the foreign substance, in recent years (Sato, T. et al., Int. Immunol., 6, 665-669, 1994). [0002]
  • MBP is a C type lectin which specifically binds to mannose, N-acetylglucosamine and the like in the presence of Ca, of which structure comprises a collagen-like domain containing at least one (Gly-Xaa-Yaa)n, and carbohydrate chain recognition domain (CRD). Similarly to MBP, lectins having a collagen-like domain and CRD are generically called as collectin (Malhotora, R. et al., Eur. J. Immunol., 22, 1437-1445, 1992), which include collectin-43 (CL-43), surfactant protein A (SP-A), surfactant protein D (SP-D), bovine conglutinin (BKg) and the like, in addition to MBP. Collectin has an opsonic activity, which is believed to participate in fundamental immunity against a variety of microorganisms such as bacteria and viruses (Kawasaki, N. et al., J. Biochem., 106, 483-489, 1989; Ikeda, K. et al., J. Biol. Chem., 262, 7451-7454, 1987; Ohta, M. et al., J. Biol. Chem., 265, 1980-1984, 1990; Summerfield, J. A. et al., Lancet, 345, 886, 1995). [0003]
  • These collectins are known to be constituted from a basic structure containing characteristic domains such as (1) CRD and (2) collagen-like domain and the like as shown in FIG. 1([0004] a) (Malhortra et al., Eur. J. Immunol., 22, 1437-1445, 1992). This basic structure forms a subunit through composing a triple helix at the collagen-like domain, and thus these subunits further form an oligomer structure such as trimer, tetramer, hexamer and the like.
  • Recently, collectins were suggested to participate in non-specific immune response, e.g., it was reported that for example, they are playing important roles in neutralizing and excluding various microorganisms in infants having insufficient maternal antibodies from mother or having specific defense systems which were insufficiently developed (Super et al., Lancet, 2, 1236-1239, 1989). Moreover, results of investigation are reported involving in roles of these collectins in the body defense system of a host, which for example, suggest that the host becomes more susceptible to infections through the lowered concentration of MBP in blood resulting from genetic mutation of MBP (Sumiya et al., Lancet, 337, 1569-1570, 1991). In addition, it was reported that serum MBP content shows a lowered level upon the failure of opsonization (Madsen, H. O. et al., Immuno genetics, 40, 37-44, 1994), whilst bacterial infections readily occur (Garred, P. et al., Lancet, 346, 941-943, 1995). Therefore, MBP can be believed to play important roles in an immune system. [0005]
  • The present inventors previously found that BKg and MBP inhibit infections by H1 and H3 types influenzae A viruses as well as a haemagglutination activity (Wakamiya et al., Glycoconjugate J., 8, 235, 1991; Wakamiya et al., Biochem. Biophys. Res. Comm., 187, 1270-1278, 1992). Thereafter, a cDNA clone encoding BKg was obtained, and the relevance between BKg and SP-D and the like has been also found (Suzuki et al., Biochem. Biophys. Res. Comm., 191, 335-342, 1993). [0006]
  • Accordingly, collectins are substances to which usefulness in the elucidation of body defense mechanisms and utilities as a biologically active substance are expected. Thus, the finding of novel molecular species belonging to this family may greatly contribute in various medical fields and biological fields in addition to the therapy of infectious diseases. [0007]
    • DISCLOSURE OF THE INVENTION
  • The object of the present invention is to provide materials that can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and for the development of reagents and drugs for the same. [0008]
  • Accordingly, aspects provided by the present invention are as described below. [0009]
  • (1) A protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 2, or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2, or a derivative or a fragment thereof; [0010]
  • (2) A base sequence set out in SEQ ID NO: 45 (corresponding to a base sequence set out in the base position 265-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2; [0011]
  • (3) A protein comprising an amino acid sequence consisting of 245 amino acids set out in the amino acid position 1-245 of SEQ ID NO: 4, or a protein consisting of an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4, or a derivative or a fragment thereof; [0012]
  • (4) A base sequence set out in SEQ ID NO: 46 (corresponding to a base sequence set out in the base position 141-875 of SEQ ID NO: 3), a base sequence encoding an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4; [0013]
  • (5) A protein comprising an amino acid sequence consisting of 159 amino acids set out in SEQ ID NO: 47 (corresponding to an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2), or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2, or a derivative or a fragment thereof; [0014]
  • (6) A base sequence set out in SEQ ID NO: 48 (corresponding to a base sequence set out in the base position 601-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2; [0015]
  • (7) The protein further comprising a sequence (Gly-Xaa-Yaa)n, wherein n represents an integer of from 1 to 50, Xaa and Yaa represent an amino acid residue, which may be the same or different, at an N-terminal of the protein according to (5); [0016]
  • (8) The protein according to (7) wherein said (Gly-Xaa-Yaa)n is selected from the group of sequences consisting of: [0017]
  • Gly-Leu-Lys-Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg—Val-Gly-Pro-Thr-Gly-Glu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly—Arg-His-Gly-Lys-Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 49 (corresponding to the amino acid position 41-112 of SEQ ID NO: 2)), [0018]
  • Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg-Val-Gly-Pro—Thr-Gly-Glu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly—Lys-Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 50 (corresponding to the amino acid position 44-112 of SEQ ID NO: 2, or the amino acid position 18-86 of SEQ ID NO: 4)) Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 51 (corresponding to the amino acid position 92-112 of SEQ ID NO: 2 or the amino acid position 66-86 of SEQ ID NO: 4)), [0019]
  • Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly-Lys-Ile-Gly-Pro—Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 52 (corresponding to the amino acid position 68-112 of SEQ ID NO: 2 or the amino acid position 42-86 of SEQ ID NO: 4)), [0020]
  • Gly-Asp-Ala-Gly-Glu-Lys-Gly.-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg-Val-Gly-Pro—Thr-Gly-Glu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 11 (corresponding to the amino acid position 44-67 and 92-112 of SEQ ID NO: 2 or the amino acid position 18-41 and 66-86 of SEQ ID NO: 4)), Gly-Leu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 43 (corresponding to the amino acid position 41-43 and 92-112 of SEQ ID NO: 2)), [0021]
  • Gly-Leu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly-Lys—Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 42 (corresponding to the amino acid position 41-43 and 68-112 of SEQ ID NO: 2)), or [0022]
  • Gly-Leu-Lys-Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg—Val-Gly-Pro-Thr-Gly-Glu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 44 (corresponding to the amino acid position 41-67 and 92-112 of SEQ ID NO: 2)); [0023]
  • (9) A base sequence encoding the protein according to (7) or (8); [0024]
  • (10) A protein comprising an amino acid sequence of: [0025]
  • Met-Arg-Gly-Asn-Leu-Ala-Leu-Val-Gly-Val-Leu-Ile-Ser-Leu-Ala-Phe-Leu-Ser-Leu—Leu-Pro-Ser-Gly-His-Pro-Gln-Pro-Ala-Gly-Asp-Asp-Ala-Cys-Ser-Val-Gln-Ile-Leu-Val-Pro (SEQ ID NO: 53 (corresponding to the amino acid position 1-40 of SEQ ID NO: 2)) at an N-terminal of the sequence (Gly-Xaa-Yaa)n of the protein according to (7) or (8) in addition thereto; [0026]
  • (11) A protein comprising a sequence of: [0027]
  • Met-Trp-Trp-Val-Pro-Pro-Ser-Pro-Tyr-Gly-Cys-Leu-Pro-Cys-Ala-Leu-Pro (SEQ ID NO: 54 (corresponding to the amino acid position 1-17 of SEQ ID NO: 4)) at an N-terminal of the sequence (Gly-Xaa-Yaa)n of the protein according to (7) or (8) in addition thereto. [0028]
  • (12) A base sequence encoding the protein according to (10) or (11); [0029]
  • (13) A protein comprising an amino acid sequence consisting of 197 amino acids set out in the amino acid position 1-197 of SEQ ID NO: 6, or a protein consisting of an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6, or a derivative or a fragment thereof; [0030]
  • (14) A base sequence set out in SEQ ID NO:55 (corresponding to a base sequence set out in the base position 141-731 of SEQ ID NO: 5), a base sequence encoding an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6; [0031]
  • (15) A protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 8, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8, or a derivative or a fragment thereof; [0032]
  • (16) A base sequence set out in SEQ ID NO: 56 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 7), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8; [0033]
  • (17) A protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 10, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10, or a derivative or a fragment thereof; [0034]
  • (18) A base sequence set out in SEQ ID NO: 57 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 9), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10; [0035]
  • (19) A protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 13, or a protein consisting of an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13, or a derivative or a fragment thereof; [0036]
  • (20) A base sequence set out in SEQ ID NO: 58 (corresponding to a base sequence set out in the base position 157-969 of SEQ ID NO: 12), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13; [0037]
  • (21) A protein comprising an amino acid sequence consisting of 223 amino acids set out in the amino acid position 1-223 of SEQ ID NO: 37, or a protein consisting of an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37, or a derivative or a fragment thereof; [0038]
  • (22) A base sequence set out in SEQ ID NO: 59 (corresponding to a base sequence set out in the base position 265-933 of SEQ ID NO: 36), a base sequence encoding an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37; [0039]
  • (23) A protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 39, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39, or a derivative or a fragment thereof; [0040]
  • (24) A base sequence set out in SEQ ID NO: 60 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 38), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39; [0041]
  • (25) A protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 41, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41, or a derivative or a fragment thereof; [0042]
  • (26) A base sequence set out in SEQ ID NO: 61 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 40), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in amino acid position 1-247 of SEQ ID NO: 41; [0043]
  • (27) A vector comprising a base sequence according to (2), (4), (6), (9), (12), (14), (16), (18), (20), (22), (24) or (26); [0044]
  • (28) A transformed cell carrying a base sequence according to (2), (4), (6), (9), (12), (14), (16), (18), (20), (22), (24) or (26) in a manner to allow the expression; [0045]
  • (29) A process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to (2), (4), (6), (9), (12), (14), (16), (18), (22), (24) or (26); and harvesting thus produced hCL-L2s protein; [0046]
  • (30) A process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to (20); and harvesting thus produced mCL-L2s protein; [0047]
  • (31) The process according to (29) or (30) wherein said cell is [0048] Escherichia coli, an animal cell or an insect cell;
  • (32) A transgenic non-human animal having an altered expression level of CL-L2s gene; [0049]
  • (33) A knockout mouse wherein a function of mCL-L2s gene is deficient; [0050]
  • (34) An antibody to the protein according to (1), (3), (5), (7), (8), (10), (11), (13), (15), (17), (19), (21), (23) or (25), or a fragment thereof; [0051]
  • (35) The antibody according to (34) , which is a polyclonal antibody, a monoclonal antibody or a peptide antibody; [0052]
  • (36) A process for measuring a CL-L2s protein or a fragment thereof on the basis of an immunological binding between the antibody according to (34) or (35) and the CL-L2s protein or a fragment thereof; [0053]
  • (37) An agonist that stimulates a function of the protein according to (1), (3), (5), (7), (8), (10), (11), (13), (15), (17), (19), (21), (23) or (25); [0054]
  • (38) An antagonist that inhibits a function of the protein according to (1), (3), (5), (7), (8), (10), (11), (13), (15), (17), (19), (21), (23) or (25); [0055]
  • (39) A process for screening a drug wherein the protein according to (1), (3), (5), (7), (8), (10), (11), (13), (15), (17), (19), (21), (23) or (25) is used; [0056]
  • (40) A drug which is obtained by the process for the screening according to (39).[0057]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a schematic drawing illustrating a basic structure (a) of principal collectins reported heretofore and an overview of the protein (MBP, SP-A and SP-D). [0058]
  • FIG. 2 is a drawing illustrating a preceding half of an alignment of amino acid sequences of four kinds of collectins reported heretofore. [0059]
  • FIG. 3 is a drawing illustrating a latter half of the alignment to that of FIG. 2. [0060]
  • FIG. 4 depicts drawings (a) and (b) showing each primer employed for the determination of the sequence of the novel collectin of the present invention, and the novel collectin obtained thereby. [0061]
  • FIG. 5 is a drawing illustrating a preceding half of an alignment of amino acid sequences of four kinds of collectins reported heretofore and the novel collectin (hCL-L2-1) of the present invention. [0062]
  • FIG. 6 is a drawing illustrating a latter half of the alignment to that of FIG. 5. [0063]
  • FIG. 7 shows results of analysis of distribution of mRNA in various human tissues demonstrating the tissue distribution of the novel collectin hCL-L2 of the present invention. [0064]
  • FIG. 8 shows a phylogenetic tree of various collecting. [0065]
  • FIG. 9 shows results of analysis of distribution of mRNA in various human tissues demonstrating the tissue distribution of the novel collectin hCL-L2-1 and hCL-L2-2 of the present invention. [0066]
  • FIG. 10 is a schematic drawing illustrating a sugar binding characteristics of the novel collectin hCL-L2-1 of the present invention.[0067]
    • BEST EMBODIMENT FOR CARRYING OUT THE INVENTION
  • The present inventors successfully cloned human and mouse novel collectin genes (hCL-L2-1 and mCL-L2). A CRD (amino acid position 113-271 of SEQ ID NOs: 2 and 13, amino acid position 87-245 of SEQ ID NO: 4), which is believed to participate in fundamental immunity, and a collagen-like domain (the amino acid position 41-112 of SEQ (ID NOs: 2 and 13, the amino acid position 18-86 of SEQ ID NO: 4) having a sequence of (Gly-Xaa-Yaa)n were present at C-terminal of the novel CL-L2. [0068]
  • Moreover, two kinds of proteins (CL-L2-1 and CL-L2-2) having different N-terminal amino acid sequences were found for hCL-L2 as shown in FIG. 4. Specifically, N-terminal amino acids of CL-L2-1 (position 1-43) set out in SEQ ID NO: 2 and N-terminal amino acids of CL-L2-2 (position 1-17) set out in SEQ ID NO: 4 were different, whilst the rest being identical. Further, although position of 1-43 of N-terminal amino acid of CL-L2-1 contained a signal sequence and one coil of a collagen-domain and the like, there were neither a signal sequence nor one coil of a collagen-like domain in position of 1-17 of N-terminal amino acid of CL-L2-2. [0069]
  • In addition, there existed three kinds of proteins corresponding to CL-L2-2, which result from alternative splicing of mRNA. Those are referred to as CL-L2-2v1 (SEQ ID NO: 5, 6), CL-L2-2v2 (SEQ ID NO: 7, 8) and CL-L2-2v3 (SEQ ID NO: 9, 10). CL-L2-2v1 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 18 to 65 (i.e., deletion of the base position of from 192 to 335 of CL-L2-2 set out in SEQ ID NO: 3); CL-L2-2v2 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 18 to 41 (i.e., deletion of the base position of from 192 to 263 of CL-L2-2 set out in SEQ ID NO: 3); and CL-L2-2v3 is a form derived from CL-L2-2 set out in SEQ ID NO: 4 with deletion of the amino acid position of from 42 to 65 (i.e., deletion of the base position of from 264 to 335 of CL-L2-2 set out in SEQ ID NO: 3). These three kinds of proteins all resulted from differential splicing of CL-L2-2 within the collagen-like domain thereof. [0070]
  • Furthermore, there existed three kinds of proteins corresponding to CL-L2-1, which result from alternative splicing of mRNA. Those are referred to as CL-L2-1v1 (SEQ ID NO: 36, 37), CL-L2-1v2 (SEQ ID NO: 38, 39) and CL-L2-1v3 (SEQ ID NO: 40, 41). CL-L2-1v1 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino acid position of from 44 to 91 (i.e., deletion of the base position of from 394 to 537 of CL-L2-1 set out in SEQ ID NO: 1); CL-L2-1v2 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino acid position of from 44 to 67 (i.e., deletion of the base position of from 394 to 465 of CL-L2-1 set out in SEQ ID NO: 1); and CL-L2-1v3 is a form derived from CL-L2-1 set out in SEQ ID NO: 2 with deletion of the amino-acid position of from 68 to 91 (i.e., deletion of the base position of from 466 to 537 of CL-L2-1 set out in SEQ ID NO: 1). These three kinds of proteins all resulted from differential splicing of CL-L2-1 within the collagen-like domain thereof. [0071]
  • hCL-L2 gene used herein includes hCL-L2-1 set out in SEQ ID NO: 1, hCL-L2-2 set out in SEQ ID NO: 3, hCL-L2-2v1 set out in SEQ ID NO: 5, hCL-L2-2v2 set out in SEQ ID NO: 7, hCL-L2-2v3 set out in SEQ ID NO: 9, hCL-L2-1v1 set out in SEQ ID NO: 36, hCL-L2-1v2 set out in SEQ ID NO: 38, and hCL-L2-1v3 set out in SEQ ID NO: 40 respectively, unless otherwise stated. hCL-L2 protein used herein includes hCL-L2-1 set out in SEQ ID NO: 2, hCL-L2-2 set out in SEQ ID NO: 4, hCL-L2-2v1 set out in SEQ ID NO: 6, hCL-L2-2v2 set out in SEQ ID NO: 8, hCL-L2-2v3 set out in SEQ ID NO: 10, hCL-L2-1v1 set out in SEQ ID NO: 37, hCL-L2-1v2 set out in SEQ ID NO: 39, and hCL-L2-1v3 set out in SEQ ID NO: 41 respectively, unless otherwise stated. In addition, homologues, variants, modified forms and polymorphic variants thereof (these are collectively referred to as “derivatives”), and fragments of the same are also included. These may be naturally occurring or artificially produced. The present invention includes any of the foregoing substances. [0072]
  • mCL-L2 gene used herein includes mCL-L2 set out in SEQ ID NO: 12, unless otherwise stated. mCL-L2 protein used herein includes mCL-L2 set out in SEQ ID NO: 13, unless otherwise stated. In addition, homologues, variants, modified forms and polymorphic variants thereof (these are collectively referred to as “derivatives”), and fragments of the same are also included. These may be naturally occurring or artificially produced. The present invention includes any of the foregoing substances. [0073]
  • Moreover, the present invention also involves amino acid sequences substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13; and base sequences encoding amino acid sequences substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13. Furthermore, proteins comprising these amino acid sequences are also involved. The amino acid sequence substantially similar to the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or to the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13 refers to the amino acid sequence having alteration such as substitution, deletion, addition and/or insertion of one or several amino acids therein as long as the protein has an equal property to that of the protein comprising an amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or the amino acid sequence set out in the amino acid position of 113-271 of SEQ ID NO: 13. These may be naturally occurring, or artificially synthesized. [0074]
  • Such deletion, substitution, and/or addition of one or several amino acids referred to herein may be deletion, substitution, and/or addition of amino acid(s) without large extent of alteration of residues having hydrophobic, hydrophobic, acidic, basic group or the like of the novel collectin, and with small extent of alteration of each fundamental characteristics of (1) Ca[0075] 2+ dependent carbohydrate chain recognition domain (CRD) and (2) collagen-like domain. On the basis of amino acid sequences and structures of proteins of collectin family reported heretofore, it is believed that deletion, substitution, and/or addition of for example, (1) from about 1 to 10 amino acids in Ca2+ dependent carbohydrate chain recognition domain (CRD) and (2) from about 1 to 50, preferably from about 1 to 15 amino acids in collagen-like domain may be allowed.
  • Equal property herein refers to a property inherent to a protein having an amino acid sequence prior to the alteration, e.g., a feature involving in tertiary structure of a protein. [0076]
  • Furthermore, the present invention also involves a nucleic acid sequence set out in SEQ ID NO: 1, 3, 5, 7, 9, 12, 36, 39 or 41, SEQ ID NO: 48 (corresponding to the base position of 601-1077 of SEQ ID NO: 1) or in the nucleic acid position of 493-969 of SEQ ID NO: 12, or a nucleic acid sequence comprising a fragment thereof, or a nucleic acid sequence that can hybridize to a nucleic acid sequence complementary thereto (hereinafter, referred to as “specified sequence”) under a stringent condition. The stringent condition according to the present invention may involve a condition for example; incubating in a solution containing 5×SSC, 5% Denhardt's solution (0.1% BSA, 0.1% Ficol 1400, 0.1% PVP), 0.5% SDS and 20 μg/ml denatured sermon sperm DNA at 37° C. overnight followed by a wash with 2×SSC containing 0.1% SDS at room temperature. SSPE may be employed in place of SSC. Thus resultant nucleic acid sequence is speculated to exhibit at least 50% or greater homology to the specified sequence. Many of the proteins encoded by a nucleic acid sequence that can hybridize to the specified sequence under a stringent hybridization condition are believed to have an equal property to CL-L2s protein. Therefore, such proteins are also involved in the present invention as long as they have an equal property to CL-L2s protein. [0077]
  • In particular, the amino acid sequence of hCL-L2-1 set out in SEQ ID NO: 2 (amino acid position 1-271) represents a protein consisting of 271 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 45) consists of 813 bases. Characteristic amino acid sequences such as those of a signal sequence, a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 1. [0078]
  • The amino acid sequence of hCL-L2-2 set out in SEQ ID NO: 4 (amino acid position 1-245) represents a protein consisting of 245 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 46) consists of 735 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 3. [0079]
  • The amino acid sequence of hCL-L2-2v1 set out in SEQ ID NO: 6 (amino acid position 1-197) represents a protein consisting of 197 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 55) consists of 591 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 5. [0080]
  • The amino acid sequence of hCL-L2-2v2 set out in SEQ ID NO: 8 (amino acid position 1-221) represents a protein consisting of 221 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 56) consists of 663 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 7 [0081]
  • The amino acid sequence of hCL-L2-2v3 set out in SEQ ID NO: 10 (amino acid position 1-221) represents a protein consisting of 221 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 57) consists of 663 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 9. [0082]
  • The amino acid sequence of hCL-L2-1v1 set out in SEQ ID NO: 37 (amino acid position 1-223) represents a protein consisting of 223 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 59) consists of 669 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 36. [0083]
  • The amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 39 (amino acid position 1-247) represents a protein consisting of 247 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 60) consists of 741 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 38. [0084]
  • The amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 41 (amino acid position 1-247) represents a protein consisting of 247 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 61) consists of 741 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 40. [0085]
  • Moreover, the amino acid sequence of mCL-L2 set out in SEQ ID NO: 13 (amino acid position 1-271) represents a protein consisting of 271 amino acids, and thus the base sequence encoding the same (SEQ ID NO: 58) consists of 813 bases. Characteristic amino acid sequences such as those of a collagen-like domain, a CRD domain and the like were present in the sequence. The base sequence encoding this protein is set out in SEQ ID NO: 12. [0086]
  • Homologues used herein refer to nucleic acid sequences or amino acid sequences that bear high homology, which are homologous at least 50% or more, preferably 70% or more, more preferably 90% or more. When deletion or insertion is present in the sequence, homologous search may be conducted which allows for gap junction. For example, the search may be performed using a procedure of multiple alignment (trade name: SODHO, Fujitsu Limited). In addition, as the algorithm for searching homology, Smith-Waterman algorithm, which is the most accurate, may be employed. Alternatively, FASTA or BLAST may be also utilized via the Internet. [0087]
  • Mutants used herein include for example, those resulting from allele, Single Nucleotide Polymorphism (SNP) and the like. Furthermore, the nucleic acid sequence of the present invention may also include the mutated nucleic acid sequences derived from the changes in the range of degeneracy of the codon. Partial alteration of the codon of a nucleic acid sequence may be achieved according to a conventional procedure using such site directed mutagenesis methods as those in which a primer is employed consisting of a synthesized oligonucleotide that encodes the desired alteration (Mark, D. F. et al., Proc. Nati. Acad. Sci. USA., 81, 5662, 1984). Thus resultant artificial genetic mutants are also involved in the nucleic acid sequence of the present invention. Furthermore, the mutated amino acid translated by the mutated codon has preferably similar properties to those of normal amino acid even in the case where the mutation is beyond the range of degeneracy of the codon. The mutation may be preferably as follows, which are among amino acids having similar properties, functions, characteristics and the like, for example: the mutation among aliphatic amino acids such as alanine, valine, leucine and isoleucine; the mutation among neutral amino acids such as glycine, alanine, serine, threonine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, proline, tryptophan, asparagines and glutamine; the mutation among acidic amino acids such as aspartic acid and glutamic acid; the mutation among basic amino acids such as arginine, lysine and histidine; the mutation among serine and threonine, having a hydroxyl group; the mutation among phenylalanine and tyrosine, having an aromatic ring; and the like. These artificially or naturally mutated proteins are also included in the protein of the present invention. For the artificial mutation, site-directed mutagenesis may be caused using a PCR method, or alternatively, other known methods may be used to cause mutation at any optional site. [0088]
  • Modified forms used herein may be prepared using conventional techniques, for example, by acetylation, acylation, ADP-ribosylation, amidation, myristoylation, glycosylation, hydroxylation, phosphorylation, sulfation, formylation, methylation, polyethyleneglycolation, lipid coupling, nucleotide coupling, metal coupling (calcium addition and the like), fusion with other protein (albumin and the like), dimerization, and the like. For example, since glycosylation does not occur when the host is in [0089] Escherichia coli, the expression may be conducted in eucaryotic cells when glycosylation is intended. Insect cells may be also used because glycosylation proceeds post-translationally in these cells, similarly to in mammalian cells.
  • Polymorphic variants used herein involve for example, polymorphisms caused by structural or conformational differences in chromosomal DNA, polymorphisms resulting from a change of a gene into its allelic gene, or the like. In general, genes of eucaryotic cells often exhibit polymorphic event, and this event may lead to the substitution of one or more amino acid(s), whilst the activity of the protein may be retained in spite of the substitution. Therefore, any of the genes encoding a protein obtained by artificially modifying the gene encoding any of the amino acid sequence set out in either SEQ ID NO: 2, 4, 6, 8, 10 or 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13 is involved in the present invention as far as the protein has a characteristic function of a gene according to the present invention. In addition, any of the proteins prepared by artificial modification of amino acid sequence set out in either SEQ ID NO: 2, 4, 6, 8, 10 or 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13 is involved in the present invention as far as it has a characteristics of a protein according to the present invention. The modification is construed as involving substitution, deletion, addition and/or insertion. [0090]
  • Fragments used herein refer to any optional fragments derived from the amino acid sequence of CL-L2s described above, which may include for example, an extracellular domain, an intracellular domain, a transmembrane domain, a collagen-like domain, a CRD domain, a collectin-like domain, a hydrophobic domain (a transmembrane domain and the like), a hydrophilic domain (domains other than hydrophobic domains), and the like, as well as fragments obtained by the fusion of these fragments. [0091]
  • For example, in the amino acid sequence of hCL-L2-1 set out in SEQ ID NO: 2, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 113 to 271 that form a CRD domain; a fragment comprising amino acids of approximately from the [0092] position 41 to 271 that form a CRD domain and a collagen-like domain; and a fragment comprising amino acids of approximately from the position 41 to 112 that form a collagen-like domain. Furthermore, in the amino acid sequence of hCL-L2-2 set out in SEQ ID NO: 4, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 245 that form a CRD domain and a collagen-like domain, and a fragment comprising amino acids of approximately from the position 18 to 86 that form a collagen-like domain; in the amino acid sequence of hCL-L2-2v1 set out in SEQ ID NO: 6, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 197 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 18 to 38 that form a collagen-like domain; in the amino acid sequence of hCL-L2-2v2 set out in SEQ ID NO: 8, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 221 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 18 to 62 that form a collagen-like domain; in the amino acid sequence of hCL-L2-2v3 set out in SEQ ID NO: 10, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 18 to 221 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 18 to 62 that form a collagen-like domain. Moreover, in the amino acid sequence of hCL-L2-1v1 set out in SEQ ID NO: 37, exemplary fragments may include: a fragment comprising amino acids of from the position approximately 41 to 223 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 64 that form a collagen-like domain; in the amino acid sequence of hCL-L2-1v2 set out in SEQ ID NO: 39, the fragments included may be: a fragment comprising amino acids of approximately from the position 41 to 247 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 88 that form a collagen-like domain; in the amino acid sequence of hCL-L2-1v3 set out in SEQ ID NO: 41, exemplary fragments may include: a fragment comprising amino acids of approximately from the position 41 to 247 that form a CRD domain and a collagen-like domain, a fragment comprising amino acids of approximately from the position 41 to 88 that form a collagen-like domain. Additionally, in the amino acid sequence of mCL-L2 set out in SEQ ID NO: 13, exemplary fragments may also include: a fragment comprising amino acids of approximately from the position 113 to 271 that form a CRD domain; a fragment comprising amino acids of approximately from the position 41 to 271 that form a CRD domain and a collagen-like domain; and a fragment comprising amino acids of approximately from the position 41 to 112 that form a collagen-like domain.
  • Process for Obtaining CL-L2s Gene [0093]
  • A CL-L2s gene according to the present invention may be those obtained through any processes. For example, the base sequence encoding CL-L2s of the present invention can be obtained by preparing mRNA from the cells that are expressing the protein, and converting it into a double stranded DNA by a conventional technique. For the preparation of mRNA, guanidine isothiocyanate calcium chloride method (Chirwin, et al., Biochemistry, 18, 5294, 1979) and the like can be employed. For the preparation of poly(A)[0094] + RNA from total RNA, supports bound with oligo(dT), for example, affinity chromatography in which sepharose or latex particles are used, can be employed. Double stranded cDNA can be obtained by using thus obtained RNA described accordingly as a template to treat with reverse transcriptase, using oligo(dT) that is complementary to poly(A) chain present at 3′-terminus, or a random primer or a synthesized oligonucleotide corresponding to a part of the amino acid sequence of CL-L2s as a primer (Mol. Cell Biol., 2, 161, 1982; Mol. Cell Biol., 3, 280, 1983; Gene, 25, 263, 1983); and by treating thus resulting cDNA strand with for example, E. coli RNaseH, E. coli DNA polymerase 1, E. coli DNA ligase to alter into the DNA strand. A cDNA library can be produced by incorporating this cDNA into a plasmid vector, a phage vector or a cosmid vector to transform E. coli, or by transfecting it into E. coli following in vitro packaging.
  • The plasmid vector that can be used herein is not particularly limited as long as it can be replicated and maintained in the host. Phage vector is not also particularly limited as long as it can proliferate in the host. Cloning vectors include, for example, pBR322, pUC19, λgt10, λgt11 and the like. Moreover, upon subjecting to immunological screening, the vector has preferably a promoter that enables the expression of a CL-L2s gene in the host. [0095]
  • To incorporate cDNA into a plasmid, the process of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition) and the like can serve as a reference. Further, to incorporate cDNA into a phage vector, the process disclosed in Hyunh et al. (DNA cloning, a practical approach, 1, 49, 1985) and the like can serve as a reference. [0096]
  • As the process for introducing the expression vector described above into host cells, methods for example, transfection by lipopolyamine method, DEAE-dextran method, Hanahan method, lipofectin method, calcium phosphate method; microinjection, and electroporation and the like (Molecular Cloning, A Laboratory Manual, second edition) may be involved. In vitro packaging can be readily effected by using commercially available kits (manufactured by Stratagene, or Amersham). [0097]
  • The process for the isolation of cDNA encoding a CL-L2s protein from a cDNA library prepared as described above may involve a general process, which may be used in combination, for the screening of cDNA. For example, a probe labeled with [0098] 32P is produced, and a clone containing the desired cDNA can be screened by a colony hybridization method (Proc. Natl. Acad. Sci. USA, 72, 3961, 1975), or a plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, 2, 108, 1989). Further, a clone may be selected by a PCR method. Additionally, the desired clone can be selected through the use of an antibody that recognizes CL-L2s when a cDNA library is produced using a vector that can express cDNA.
  • Furthermore, when a CL-L2s gene is isolated from cells that express CL-L2s gene, for example, the expressing cells are dissolved using SDS or proteinase K, followed by a phenol treatment. Unwanted RNA is digested with ribonuclease. Thus resultant DNA is digested with restriction enzyme, and the resulting DNA fragments are amplified using phage or cosmid to produce a library. Thereafter, the desired clone is selected, and then a CL-L2s gene can be obtained. [0099]
  • The base sequence of the DNA obtained accordingly can be determined by a Maxam-Gilbert method (Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or a Sanger's method (Proc. Nati. Acad. Sci. USA, 74, 5463, 1977). The CL-L2s gene can be obtained by excising from the clone as obtained above. [0100]
  • Through use of the primer synthesized on the basis of the base sequence of CL-L2, cloning can be also effected by a RT-PCR method using poly(A)[0101] + RNA of the cells expressing CL-L2s as a template. Further, the desired cDNA can be also obtained by directly screening the cDNA library after producing/synthesizing a probe based on the base sequence of CL-L2s, not by way of the PCR. The gene of the present invention can be selected among the genes obtained by the methods described herein above through the verification of the base sequence of the gene. The gene of the present invention can be also produced according to the conventional process in which chemical synthesis of a nucleic acid, e.g., phosphoimidite method (Mattencci, M. D. et al., J. Am. Chem. Soc., 130, 3185, 1981) or the like, is employed.
  • Process for Producing Expression Vector [0102]
  • The present invention also relates to a vector comprising a nucleic acid sequence of CL-L2s. The vector is not particularly limited so far as it can express the CL-L2s protein, however, a plasmid vector, an RNA vector, a DNA vector, a virus vector, a phage vector and the like may be employed. Specifically, examples thereof include pBAD/His, pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5, pcDNA3.1 or pSecTag2 manufactured by Invirtogen, pET or pBAC manufactured by Novagen Co., pGEM manufactured by Promega, pBluescriptII, pBs, Phagescript, pSG or pSV2CAT manufactured by Stratagene, or pGEX, pUC18/19, pBPV, pSVK3 or pSVL manufactured by Pharmacia Co. [0103]
  • The cDNA sequence of CL-L2s ligated to the expression vector is operatively linked to a promoter. The promoter includes for example, phage λPL promoter, [0104] E.coli lac, trp, tac promoter, SV40 early and late promoter, T7 and T3 promoter, retrovirus LTR promoter. Specifically, the prompter for use in eukaryotic cells include CMV promoter, HSV promoter, SV40 early and late promoter, retrovirus LTR promoter, RSV promoter, metallothionein promoter. In addition, the expression vector may contain a marker to allow the selection of the transformed host, and an enhancer. Examples of the marker include dihydrofolate reductase gene, neomycin resistant gene, ampicillin resistant gene and the like. Examples of the enhancer include SV40 enhancer, cytomegalovirus early enhancer promoter, adenovirus enhancer and the like.
  • Process for Producing Transformed Cells [0105]
  • The present invention further provides transformed cells carrying a base sequence of the present invention to allow the expression thereof by means of the vector as described above that is carrying the base sequence. The host cell for use as a transformed cell in the present invention may preferably include animal cells and insect cells, however, included may be all of the cells (microorganisms may be also included), which can express a CL-L2s protein in the expression vector of the present invention. [0106]
  • Exemplary animal cells or insect cells of the present invention may respectively include cells derived from human, or cells derived from fly or silkworm (Bombyx mor). For example, CHO cells, COS cells, BHK cells, Vero cells, myeloma cells, HEK293 cells, HeLa cells, Jurkat cells, mouse L cells, mouse C127 cells, mouse FM3A cells, mouse fibroblast, osteoblast, chondrocyte, S2, Sf9, Sf21, High Five™ cells may be included. The microorganism according to the present invention include [0107] Escherichia coli, Saccharomyces cerevisiae and the like. For the introduction of a vector into such hosts, the method as described above may be employed.
  • The cells expressing CL-L2s of the present invention can be used for analyzing a collectin pathway involving in infectious diseases, immunity and the like. Further, those cells can be utilized in manufacturing a CL-L2s protein or a CL-L2s protein having a carbohydrate chain. The cells can also be utilized in screening for the purpose of obtaining an agonist or an antagonist to the CL-L2s protein. [0108]
  • Process for Obtaining Protein [0109]
  • The present invention also relates to a process for the production of a CL-L2s protein which comprises culturing a cell transformed with the base sequence of the present invention as set forth above, and harvesting thus produced CL-L2s. Cell culture, isolation of the protein, and purification thereof may be carried out with conventionally known processes. [0110]
  • The protein according to the present invention can be expressed as a recombinant fusion protein, which can be readily isolated, purified, and recognized per se. The recombinant fusion protein is a protein expressed by adding an appropriate peptide chain to the N-terminal end and/or C-terminal end of a protein expressed from a nucleic acid sequence encoding the desired protein. In order to facilitate the purification of the expressed protein, the protein may be expressed as a fusion protein having a signal for extracellular secretion. In addition, the protein can be obtained from several kinds of sources such as cultured cells, cultured tissues, transformed cells and the like using conventionally known methods, for example, known purification methods including: salting out such as ammonium sulfate precipitation technique and the like; gel filtration technique using Sephadex and the like; ion exchange chromatographic technique; hydrophobic chromatographic technique; dye gel chromatographic technique; electrophoresis technique; dialysis; ultrafiltration technique; affinity chromatographic technique; high performance liquid chromatographic technique; and the like. [0111]
  • Process of the Utilization of Gene [0112]
  • Probes for detecting CL-L2s gene can be specified on the basis of the base sequence set out in either SEQ ID NO: 1, 3, 5, 7, 9, 12, 36, 38 or 40, SEQ ID NO: 48 (corresponding to the base position of 601-1077 of SEQ ID NO: 1) or in the nucleic acid position of 493-969 of SEQ ID NO: 12. Alternatively, primers can be specified for the amplification of DNA or RNA including such a base sequence. To specify a probe or a primer based on a given sequence is ordinarily carried out by those skilled in this art. An oligonucleotide having a specified base sequence can be obtained through chemical synthesis. When a suitable. label is added to the oligonucleotide, it can be utilized for hybridization assay in several formats. Alternatively, it can be also utilized in reactions for synthesis of nucleic acids such as PCR. The oligonucleotide that is utilized as a primer is of at least 10 bases in length, and suitably of 15 to 50 bases in length. It is desirable that the oligonucleotide that is used as a probe be of from 100 bases to its full length. Further, they can be also used for the diagnosis of diseases caused by mutation of a CL-L2s gene because they can be used for detecting genetic mutation encoding a CL-L2s protein and for detecting SNP. They are expected to be available for the diagnosis of a variety of diseases including for example, bacterial infections and the like. In addition, they are also useful for gene therapy whereby CL-L2s gene is introduced into a living body to allow the expression thereof. [0113]
  • Moreover, it is also possible to obtain a promoter region and an enhancer region of the CL-L2s gene that is present in a genome, based on a cDNA base sequence of CL-L2s provided by the present invention. In particular, these control regions can be obtained by similar methods to those disclosed in Japanese unexamined patent publication No. 6-181767; J. Immunol., 155, 2477, 1995; Proc. Nati. Acad. Sci, USA., 92, 3561, 1995, and the like. Promoter region referred to herein means a DNA region which controls the expression of a gene that exists upstream of a transcription initiation site. Enhancer region herein refers to a DNA region that enhances the expression of a gene that exists in an intron, a 5′-untranslated region, or a 3′-untranslated region. [0114]
  • Process of the Utilization of Protein [0115]
  • CL-L2s proteins of the present invention can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and for the development of reagents and drugs for the same. Furthermore, they can be used as an antigen for producing antibodies to CL-L2s. Additionally, they can be utilized in the screening processes of an agonist or an antagonist. [0116]
  • Agonist and Antagonist [0117]
  • The present invention also relates to agonists which stimulate the activity or the activation of CL-L2s of the present invention. In addition, the present invention also relates to antagonists which inhibit the activity or the activation of CL-L2s of the present invention. For screening the antagonist, a competitive experimental system can be used, for example, in which mannose or an antibody, and a candidate inhibitor are subjected to the interaction with cells expressing CL-L2s protein thereby allowing the candidate inhibitor to screen based on the binding ratio to mannose. Otherwise, conventionally known methods may also be carried out to effect the screening. Further, the antagonists also include antisense nucleic acids that inhibit the expression of CL-L2s gene. Included in the examples of the other methods for the screening may be methods in which a change in extracellular pH is measured, which is caused by the activation of a receptor (Science, 246, 181-296, 1989), and the like. [0118]
  • Transgenic Non-human Animal [0119]
  • The present invention relates to transgenic non-human animals having an altered expression level of CL-L2s gene. CL-L2s gene herein includes cDNA, genomic DNA or synthesized DNA encoding hCL-L2s or mCL-L2s. For expression of a gene, any one of the steps of transcription and translation should be comprised. The transgenic non-human animals according to the present invention are useful for the investigation of functions or expression mechanisms of CL-L2, elucidation of mechanisms of diseases that are anticipated to be involved in CL-L2s, development of diseased animal models for use in screening and safety tests of pharmaceutical products. [0120]
  • In the present invention, the gene can be artificially modified to increase or decrease the expression level in comparison with the native expression level of the gene by introducing mutation such as deletion, substitution, addition and/or insertion into a part of some key sites (enhancer, promoter, intron or the like) that regulate the expression of the gene to be proper. The introduction of the mutation can be carried out by known methods to obtain a transgenic animal. [0121]
  • Transgenic animals in their narrow means refer to animals having germ cells into which a foreign gene was artificially introduced by a genetic recombination technique. In their broader means, they include: antisense transgenic animals having a particular gene of which function was suppressed using an antisense RNA; knockout animals having a particular gene knocked out using embryonic stem cells (ES cell); and animals having point mutation of DNA introduced, all of which are animals having a chromosome with a foreign gene being stably introduced therein at an early stage of the development of the individual, and having a genotype that can be transmitted to the progeny thereof. [0122]
  • Transgenic animals referred to herein should be comprehended in their broader means including all vertebrates other than human. The transgenic animals according to the present invention are useful for the investigation of functions or expression mechanisms of CL-L2s, elucidation of mechanisms of diseases that are involved in cells expressed in human, development of diseased animal models for use in screening and safety tests of pharmaceutical products. [0123]
  • Process for producing a transgenic mouse may include: a process in which a gene is directly introduced into a nucleus of an ovum in a anterior nucleus phase with a micropipette under a phase contrast microscope (microinjection technique, U.S. Pat. No. 4,873,191); a process in which embryonic stem cells (ES cells) are used. Alternatively, a process in which a gene is introduced into a retrovirus vector or an adenovirus vector followed by infection into an ovum; a sperm vector technique in which a gene is introduced into an ovum via a sperm; and the like have been developed. [0124]
  • The sperm vector technique is a genetic recombinant process in which a foreign gene is attached to a sperm, or a foreign gene is introduced into a sperm cell with an electroporation technique, and then the foreign gene is introduced into an ovum by fertilizing the ovum (M. Lavitranoet et al., Cell, 57, 717, 1989). Alternatively, site directed genetic recombination in vivo may be also employed by a cre/loxP recombinase system of bacteriophage P1, a FLP recombinase system of [0125] Saccharomyces cerevisiae, or the like. Additionally, a process has been also reported in which a transgene of a desired protein is introduced into a non-human animal using retrovirus.
  • Process for the production of a transgenic animal with a microinjection technique is carried out as described below, for example. [0126]
  • First, a transgene is required, which is substantially constituted from a promoter involved in expression control, a gene encoding a specified protein, and a poly(A) signal. The manner of the expression and/or the expression level of a specified molecule may be affected by the promoter activity. In addition, because transgenic animals are different among the produced lineages in respect to the number of the copies of the introduced transgene, or the introduced site in the chromosome, the manner of the expression and/or the expression level must be confirmed for each of the lineages. Since it has been elucidated that the expression level is altered depending on the untranslated region or splicing, an intron sequence to be spliced at a preceding site of poly (A) signal may be previously introduced. It is important to use a gene, which is introduced into a fertilized ovum, has as high purity as possible. The animal to be used may include mice for use in collecting fertilized ova (5-6 weeks old), male mice for use in mating, female pseudopregnant mice, vas deferens ligated male mice, and the like. [0127]
  • In order to efficiently obtain the fertilized ova, gonadotropin or the like may be used for inducing the ovulation. The fertilized ova are harvested, and thereafter, a gene in an injection pipette is introduced into a male pronucleus of the ovum by a microinjection technique. An animal (a pseudopregnant mouse or the like) for use in repositioning the injected ova to an oviduct is provided, to which 10-15 ova are transplanted per one animal. Thereafter, the born mouse can be examined for the introduction of the transgene by: extracting genomic DNA from the end portion of the tail; and detecting the transgene by a Southern method or a PCR technique, alternatively by a positive cloning technique where a marker gene is inserted which is activated upon only the occurrence of homologous recombination. Moreover, in order to ascertain the expression of the transgene, a transcription product derived from the transgene is detected by a Northern method or a RT-PCR technique. Alternatively, a western blotting method may be carried out with a specific antibody to the protein or a fragment thereof. [0128]
  • Knockout Mouse [0129]
  • The knockout mouse according to the present invention is one that was treated in a manner to deprive the function of CL-L2s gene. Knockout mouse refers to a transgenic mouse in which an arbitrary gene is destroyed by a homologous recombination technique to impair the corresponding function. The knockout mouse can be produced by homologous recombination using ES cells, followed by the selection of the embryonic stem cell having one of the allelic gene altered/destroyed. A chimeric mouse, which carry cells derived from the embryonic stem cells and cells derived from the embryo being mixed, may be obtained by, for example, injecting the embryonic stem cell that had been genetically engineered at blastocyst stage or morulae stage of the fertilized ovum. When this chimeric mouse (chimera refers to a single individual built-up with somatic cells on the basis of more than two fertilized ova) is crossbred with a normal mouse, a heterozygotic mouse can be produced having one of the allelic gene is entirely altered/destroyed. Further, a homozygotic mouse can be produced by crossbreeding heterozygotic mice each other. [0130]
  • Homologous recombination refers to the recombination that is caused by a mechanism of genetic recombination between two genes having identical or extremely similar base sequences. For the selection of cells with the homologous recombination, PCR can be employed. PCR reaction, in which primers corresponding to a part of the inserted gene and a part of the region expected to be inserted are used, may be carried out to reveal the homologous recombination occurring in cells that could yield the amplification products. Also, when the homologous recombination is caused to a gene expressed in embryonic stem cells, the gene to be introduced may be joined to a neomycin resistant gene to allow the selection after the introduction into cells by making them resistant to neomycin. Accordingly, known methods and the modified methods thereof can be employed to enable the easy selection. [0131]
  • Process for Producing Antibodies [0132]
  • The present invention further provides antibodies that recognize CL-L2s or fragments thereof. The antibodies in accordance with the present invention include for example, the antibodies to a protein comprising an amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13, or a fragment thereof. The antibodies (e.g., polyclonal antibodies, monoclonal antibodies, peptide antibodies) or antisera to CL-L2s or a fragment thereof can be produced using CL-L2s or a fragment thereof of the present invention as an antigen according to any process for producing the antibodies or antisera which is known per se. In particular, antibodies that can control the function of CL-L2s (e.g., antibodies that recognize CRD, a collagen like domain and the like) are useful for pharmaceutical products containing the antibody. [0133]
  • CL-L2s or a fragment thereof according to the present invention may be administered neat or with a diluent or a carrier to a warm-blooded animal at a site that enables the production of the antibody upon the administration. In order to facilitate the production of antibodies upon the administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration may be usually conducted once per 1 to 6 weeks, and two to ten times in total. The warm-blooded animal used may include for example, monkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat, chicken, and the like. Among these, mouse and rat may be preferably used. Rat that may be preferably used includes Wistar and SD strain rat, and mouse that may be preferably used includes BALB/c, C57BL/6 and ICR strain mouse and the like. [0134]
  • Upon the production of cells that produce a monoclonal antibody, an individual with the antibody titer that can be recognized therein is selected from the warm-blooded animals e.g., mice that had been immunized with an antigen. On two to five days after final immunization, spleen or lymph node is collected, and the antibody producing cells contained therein are subjected to the fusion with myeloma cells to effect the preparation of monoclonal antibodies producing cells. The determination of the antibody titer in the antiserum may be carried out for example, by subjecting a labeled CL-L2s described below to a reaction with the antiserum, and thereafter measuring the activity of the label bound to the antibody. The fusion operation can be performed in accordance with a known technique for example, the process of Köhler and Milstein (Nature, 256, 495, 1975) and the modified process thereof (J. Immunol. Method, 39, 285, 1980; Eur. J. Biochem., 118, 437, 1981; Nature, 285, 446, 1980). Examples of the fusion accelerating agent may include polyethylene glycol (PEG), Sendai virus and the like, and polyethylene glycol may be preferably used. In addition, lectin, poly-L-lysine or DMSO may be added ad libitum to raise the efficiency of the fusion. [0135]
  • Examples of the myeloma cell include X-63Ag8, NS-1, P3U1, SP2/0, AP-1 and the like, and SP2/0 may be preferably used. The ratio of antibody producing cell (spleen cell) number to myeloma cell number preferably used is 1:20-20:1. PEG (preferably, PEG1000- PEG6000) is added at approximately 10-80% . The fusion mixture is incubated at 20-40° C., preferably at 30-37° C. for 1-10 min. Such a condition enables efficient cell fusion. Screening of the hybridoma that produces anti-CL-L2s antibody may be performed by using various methods, which include for example, a process in which a supernatant of hybridoma culture is added to a solid phase (e.g., a microplate) adsorbed with CL-L2s antigen directly or with a carrier, and then an anti-immunoglobulin antibody (when the cells used for the cell fusion was derived from a mouse, anti-mouse immunoglobulin antibody may be used) that was labeled with a radioactive substance, enzyme or the like, or protein A is added thereto thereby detecting the anti-CL-L2s antibody bound to the solid phase; or a process in which a supernatant of hybridoma culture is added to a solid phase adsorbed with an anti-immunoglobulin antibody or protein A, and then CL-L2s labeled with a radioactive substance, enzyme or the like is added thereto thereby detecting the anti-CL-L2s monoclonal antibody bound to the solid phase. [0136]
  • Selection and cloning of the anti-CL-L2s antibody can be carried out by known methods per se, or the modified methods thereof. Usually, the process is carried out in a medium for animal cells added with HAT (hypoxanthine, aminopterin, thymidine). The medium for use in the selection, cloning and growing may be any one of the media in which hybridoma can grow. For example, RPMI medium containing 1-20%, preferably 10-20% of fetal bovine serum, GIT medium containing 1-10% of fetal bovine serum, or serum free medium for hybridoma culture, and the like. The temperature of the culture may be preferably about 37° C. The culture period may be usually five days to three weeks, preferably one week to two weeks. The culture is usually conducted in the presence of 5% carbon dioxide gas. The antibody titer of the supernatant of the hybridoma culture can be measured in a similar manner to the measurement of the antibody titer of anti-CL-L2s antibody in an antiserum as described above. In other words, a radioimmunoassay (RIA) technique, an enzyme linked immunosorbent assay (ELISA) technique, a FIA (fluorescent immunoassay) technique, a plaque measurement technique, an agglutination reaction technique and the like may be employed as the measurement process, however, the ELISA technique as described below is preferred. [0137]
  • The screening by an ELISA technique can be carried out in accordance with the following procedure. A protein, which was prepared by a similar process to that for the immunoantigen, is immobilized on the surface of each well of an ELISA plate. Next, BSA, MSA, OVA, KLH, gelatin or skimmed milk or the like is immobilized for the purpose of preventing non-specific adsorption. To each well of this plate added with a supernatant solution of the hybridoma culture, followed by allowing the immunoreaction by standing for a predetermined time. Each well is washed using a washing solution such as PBS or the like. Surfactant may be preferably added to this washing solution. An enzyme-labeled secondary antibody is added, and the mixture is allowed to stand for a predetermined time. The enzyme for labeling which can be used includes β-galactosidase, alkaline phosphatase, peroxidase and the like. After the washes with the same washing solution, enzyme reaction is effected through adding a substrate solution of the labeled enzyme that was employed. When the desired antibody is present in the added supernatant solution of the hybridoma culture, the enzyme reaction proceeds to change the color of the substrate solution. [0138]
  • Cloning can be usually carried out by known methods per se, such as a semisolid agar technique, a limiting dilution technique or the like. Specifically, after the well in which the desired antibody is produced is confirmed by the process described above, a single clone is obtained through conducting the cloning. The process for cloning may involve a limiting dilution technique or the like, in which hybridoma cells are diluted so that one colony per one well of a culture plate is formed, and thereafter the culture is conducted. Cloning by a limiting dilution technique may be performed through the use of feeder cells in order to elevate the colony formation ability, otherwise, a cell growth factor such as [0139] interleukin 6 may be added thereto. Alternatively, FACS and single cell manipulation techniques can be employed for the cloning. The cloned hybridoma is cultured preferably in a cell free medium, and an appropriate amount of the antibody is added to the supernatant thereof. Thus resulting single hybridoma may be subjected to a large scale culture using a flask or a cell culture equipment, or may be cultured in the peritoneal cavity of an animal (J. Immunol. Meth., 53, 313, 1982) to give a monoclonal antibody. When the culture is conducted in a flask, a medium for cell culture (IMDM, DMEM, RPMI 1640, MEM and the like) containing 0-20% of FCS can be used. When the culture is conducted in the peritoneal cavity of an animal, an animal of the same species, and the same strain as the animal from which myeloma cells derived that were used for the cell fusion; otherwise an athymic nude mouse may be preferably used. Hybridoma is transplanted after mineral oil such as pristine or the like is previously administered to the animal. Ascites containing the monoclonal antibody can be obtained after one to two weeks passed, when the myeloma cells enough proliferate.
  • The monoclonal antibody of the present invention can be obtained as the antibody, which does not cross-react with other proteins, by selecting one which recognizes an epitope specific for CL-L2s. In general, an epitope, which is presented by serial amino acid residues of at least more that or equal to five, preferably 7 to 20 amino acids among the amino acid sequence constituting the protein, is referred to as an epitope inherent in the protein. Therefore, the monoclonal antibody that recognizes an epitope constituted from a peptide having an amino acid sequence, which were selected from the protein comprising an amino acid set out in any of SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, SEQ ID NO: 47 (corresponding to the amino acid position of 113-271 of SEQ ID NO: 2), or in the amino acid position of 113-271 of SEQ ID NO: 13, and consisting of at least five serial amino acid residues may be identified as the monoclonal antibody specific to hCL-L2s or mCL-L2s according to the present invention. When an amino acid sequence is chosen which is conserved among the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10, 13, 37, 39 or 41, an epitope common to CL-L2s can be selected. Alternatively, a monoclonal antibody can be selected which enables the discrimination of each protein, with a region including an amino acid sequence specific for each of the sequences. [0140]
  • The separation and purification of anti-CL-L2s monoclonal antibody can be carried out according to the separation and purification process of an immunoglobulin similarly to the usual separation and purification process of the polyclonal antibodies. Known purification process which can be adopted may include for example, a salt precipitation technique, an alcohol precipitation technique, an isoelectric point precipitation technique, an electrophoretic technique, an ammonium sulfate precipitation technique, an adsorption/desorption technique by an ion exchanger (e.g., DEAE), an ultracentrifugation technique, a gel filtration technique, and a specific purification technique in which an antibody alone is collected by an antigen-bound solid phase or an active adsorbent such as protein A or protein G, or the like, followed by dissociation of the binding to give the antibody. For the purpose of preventing the formation of aggregates, or the decrease in the antibody titer in the purification step, for example, human serum albumin may be added at a concentration of 0.05-2%. Otherwise, amino acids such as glycine, α-alanine and the like, in particular, basic amino acid such as lysine, arginine, histidine and the like, saccharides such as glucose, mannitol and the like, salts such as sodium chloride may be also added. In the case of IgM, which is known to be liable to agglutinate, it may be treated with β-propionolactone and acetic anhydride. [0141]
  • The polyclonal antibody according to the present invention can be produced by known methods per se, or the modified methods thereof. For example, to produce a polyclonal antibody, an immunoantigen (a protein antigen) itself or a complex, which was formed with the immunoantigen and a carrier protein, is used for the immunization of a warm-blooded animal in a similar manner to the process for producing the monoclonal antibody described above, followed by collecting the preparation containing the antibody to the protein of the present invention or a fragment thereof from the warm-blooded animal, and then the antibody is purified/isolated. In respect to the complex of an immunoantigen and a carrier protein for use in the immunization of the warm-blooded animal, the kind of the carrier protein and the mixing ratio of the carrier and hapten may be optionally determined as long as the antibody can be efficiently produced to the hapten subjected to the immunization after crosslinking with the carrier. Thus, any kind of the carrier protein may be crosslinked at any ratio, however, the process in which about 0.1-20, preferably about 1-5 of bovine serum albumin, bovine thyroglobulin, hemocyanin or the like, for example, is coupled with 1 of hapten by weight may be used. In addition, various condensing agents may be used for the coupling of the hapten and carrier, which may include glutaraldehyde and carbodiimide, and active ester reagents containing maleimide active ester, thiol group, dithiopyridyl group and the like. The condensation product is administered neat or with a carrier or a diluent to a warm-blooded animal at a site that enables the production of the antibody upon the administration. In order to facilitate the production of antibodies upon the administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration may be usually conducted once per 2 to 6 weeks, and three to ten times in total. Polyclonal antibodies can be collected from the blood, ascites and the like, and preferably from the blood, of the warm-blooded animal immunized by a process as described above. The measurement of the antibody titer in antiserum can be carried out in a similar manner to the measurement of the antibody titer of the antiserum as described above. The separation and purification of the polyclonal antibody can be carried out according to the separation and purification process of an immunoglobulin similarly to the separation and purification process of a monoclonal antibody described above. [0142]
  • Process of the Utilization of Antibody [0143]
  • Monoclonal antibodies and polyclonal antibodies to CL-L2s or a fragment thereof can be utilized in diagnosis and therapy of the diseases relating to the cells that are expressing CL-L2s. CL-L2s or a fragment thereof can be measured using these antibodies, on the basis of the immunological binding with CL-L2s or the fragment thereof according to the present invention. Specifically, the process for measuring CL-L2s or a fragment thereof using such an antibody may include for example, sandwich techniques in which a sandwich complex is detected which was produced by subjecting CL-L2s or a fragment thereof to a reaction with an antibody coupled to an insoluble support and a labeled antibody; or competitive techniques in which CL-L2s or a fragment thereof in a sample is measured by subjecting labeled CL-L2s, and CL-L2s or a fragment thereof in a sample to a competitive reaction with the antibody followed by the measurement of CL-L2s or a fragment thereof in a sample from the amount of the labeled antigen that reacted with the antibody. [0144]
  • Upon the measurement of CL-L2s or a fragment thereof by the sandwich technique, two-step methods in which CL-L2s or a fragment thereof is first subjected to a reaction with an immobilized antibody; thereafter, unreacted materials are completely removed by washes; and then a labeled antibody is added thereto to have the immobilized antibody-CL-L2s labeled antibody formed, alternatively, one-step methods in which an immobilized antibody, a labeled antibody and CL-L2s or a fragment thereof are mixed concurrently. [0145]
  • Insoluble support for use in the measurement include for example, synthetic resin such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylic acid ester, nylon, polyacetal, fluorine-contained resin and the like; polysaccharides such as cellulose, agarose and the like; glasses; metals; and the like. The insoluble support may be in a variety of forms, and for example, tray-like, spherical, fibrous, cylindrical, discal, vessel-like, cell-like, tubular, and the like may be adopted. The support onto which the antibody had been adsorbed may be stored ad libitum in cold, in the presence of an antiseptic agent such as sodium azide and the like. [0146]
  • For the immobilization of the antibody, known chemical coupling processes or physical adsorption processes may be adopted. Chemical coupling process includes for example, processes in which glutaraldehyde is used; maleimide processes in which N-succinimidyl-4-(N-maleimidemethyl) cyclohexane-1-carboxylate and N-succinimidyl-2-maleimide acetate and the like are used; carbodiimide processes in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and the like is used. Other process includes maleimidebenzoyl-N-hydroxysuccinimide ester processes, N-succimidyl-3-(2-pyridylthio) propionic acid processes, bisdiazobenzidine processes, dipalmityl lysine processes. Alternatively, a complex that had been formed previously by subjecting the substance to be detected to a reaction with two kinds of antibodies of which epitopes are different can be captured by the third antibody to the antibody, which had been immobilized in a similar manner to those described above. [0147]
  • The material to be used for labeling may include enzyme, fluorescent materials, luminescence materials, radioactive materials, metal chelates and the like. Examples of enzyme may include peroxidase, alkaline phosphatase, β-D-galactosidase, malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, α-glycerolphosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, asparaginase, glucose oxidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase and the like. Fluorescent materials may include for example, fluorescein isothiocyanate, phycobilin protein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, orthophthalic aldehyde and the like. Luminescence materials may include isoluminol, lucigenin, luminol, aromatic acridinium esters, imidazole, acridinium salts and modified esters thereof, luciferin, luciferase, aequorin and the like. Radioactive materials may include [0148] 125I, 127I, 131I, 14C, 3H, 32P, 35S and the like. These materials are not limited thereto as long as the material can be used in immunological determination methods. In addition, low molecular weight hapten such as biotin, dinitrophenyl, pyridoxal or fluorescamine may be conjugated to the antibody. Preferably, horseradish peroxidase may be used as a labeling enzyme. This enzyme can react with many kinds of substrates, which can be readily conjugated to the antibody by a periodic acid method.
  • When an enzyme is used as a labeling agent, a substrate for measuring its activity, and a color-developing agent as needed may be employed. When peroxidase is used as an enzyme, H[0149] 2O2 may be used as a substrate solution, and 2,2′-azino-di-[3-ethylbenzthiazolin sulfonate] ammonium (ABTS), 5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine, 3,3′,5,5′-tetramethylbenzidine or the like may be used as a color-developing agent. When alkaline phosphatase is employed as an enzyme, orthophenylphosphate, paranitrophenylphosphate or the like may be used as a substrate. Alternatively, when β-D-galactosidase is used as an enzyme, fluorescein-di-(β-D-galactopyranoside), 4-methyl-umbelliferyl-β-D-galactopyranoside, or the like may be used as a substrate. The present invention also involves kit products including a monoclonal antibody, a polyclonal body described above, and reagents.
  • Available crosslinking agents include N,N′-orthophenylenedimaleimide, 4-(N-maleimidemethyl) cyclohexanoyl N-succinimide ester, 6-maleimidehexanoyl N-succinimide ester, 4,4′-dithiopyridine, and other known crosslinking agents. The reaction of such a crosslinking agent with the enzyme and the antibody may be conducted in accordance with known methods depending upon the properties of the respective crosslinking agents. Additionally, the antibodies to be used may be any fragments of the antibodies for example, Fab′, Fab, F(ab′)[0150] 2 depending on the condition. Furthermore, enzymatically labeled antibodies may be prepared by using a similar process to any one of those for polyclonal antibodies and monoclonal antibodies. When the enzymatically labeled antibody that was obtained by using the aforementioned crosslinking agent is purified by any known methods such as affinity chromatography or the like, more sensitive immunological determination system can be achieved. The enzymatically labeled antibody, which was purified in such a manner, is stored in a cold and dark place after adding thimerosal, glycerol or the like as a stabilizer, alternatively, after being lyophilized.
  • The subject sample for the measurement may be a sample containing CL-L2s, which may include body fluids such as plasma, serum, blood, urine, tissue fluid, cerebrospinal fluid and the like, various types of cells, tissues, and the like. [0151]
  • Process for Producing Humanized Antibody [0152]
  • It is ethically impermissible to produce antibodies by immunizing human with an optional antigen. Further, when a mouse monoclonal antibody is administered to a human body, there is a risk of the occurrence of a variety of adverse effects, because the antibody is a heterogeneous protein to human. Therefore, an antibody with lowered antigenicity to human is preferred when the antibody is administered to human. [0153]
  • Process for the production of human monoclonal antibodies involves transformation techniques with Epstein-Barr virus (EBV), and fusion techniques in which thus transformed cells and parent cells are fused; processes in which a chimeric antibody or a humanized antibody is produced using genetic engineering techniques; and the like in addition to cell fusion techniques. Chimeric antibody refers to an antibody that was produced by linking immunoglobulin gene fragments from heterogeneous animals. Humanized antibody refers to an antibody having a substituted primary structure in part other than a complementarity determining region (CDR) of H chain and L chain with the corresponding primary structure of a human antibody through introducing the alteration to a mouse antibody or the like that is heterogeneous to human. [0154]
  • For the production of a chimeric antibody, a mouse is immunized first, and an antibody variable region (V region) that binds to an antigen is excised from a gene of the mouse monoclonal antibody. Thereafter, the V region is linked to a gene of an antibody constant region (C region) derived from human myeloma to give a chimeric gene. Upon expression of this chimeric gene in a host cell, human-mouse monoclonal antibody can be produced. Because chimeric antibodies are less antigenic to human, they can be utilized as a monoclonal antibody for therapeutic use to be administered into a human body, or for use in diagnostic imaging. Known techniques relevant to chimeric antibodies involve Japanese patent unexamined publication No. Hei 05-304989, Japanese patent unexamined publication No. Hei 04-330295, WO9106649, Japanese patent unexamined publication No. Sho 63-036786, Japanese patent examined publication No. Hei 06-98021, and the like. [0155]
  • Moreover, humanized antibodies were recently developed, which are appreciated as being more useful than chimeric antibodies. Humanized antibody refers to an antibody that is humanized as a whole molecule except for CDR of an antibody molecule by grafting only a sequence of a gene for an antigen-binding site (CDR: complementarity determining region) of an antibody molecule into a gene of a human antibody (CDR grafting). This antibody is appreciated as being safer with less antigenicity than the human-mouse chimeric antibody because it has less part derived from a mouse antibody. When SHM-D 33 strain (ATCC CRL 1668) or RF-S1 strain, both of which being human/mouse heteromyeloma, is used as a parent cell for producing a human monoclonal antibody, high fusion efficiency can be achieved that is equivalent to mouse parent cells. Hybridoma that was obtained using these parent cells can be cloned without feeder cells, and it can produce IgG type antibody in a comparatively stable manner at a large amount. For the culture of the parent cells, ERDF medium supplemented with 15% FCS may be used, although other operation may be similarly carried out to the operation for the murine cells. Additionally, in order to produce an IgG type human monoclonal antibody, human lymphocytes collected from peripheral blood may be preferably employed, which were sufficiently sensitized with an antigen. When it is difficult to obtain sufficiently sensitized lymphocytes, sensitization with an antigen may be also conducted in vitro. In Japan, clinical trials have been currently carried out for humanized antibodies to adult T cell leukemia. In respect to the production of human antibodies and the related art, for example, reference should be made to those disclosed in Genentech Inc., USA (WO9222653, WO9845332, WO9404679, WO9837200, WO9404679) and Celltech Inc., England (WO9429451, WO9429351, WO9413805, WO9306231, WO9201059, WO9116927, WO9116928, WO9109967, WO8901974, WO8901783), and the like. [0156]
  • Using the methods and the like described above, the antibodies according to the present invention can be humanized, and such antibodies would be extremely useful upon the administration to human. [0157]
  • Composition [0158]
  • The CL-L2s polynucleotides or proteins are possibly utilized in diagnostic, prophylactic and therapeutic methods, and for the development of reagents and drugs for various types of diseases including bacterial infections and the like. [0159]
  • Pharmaceutical composition according to the present invention may comprise CL-L2s polynucleotides or proteins, substances that stimulate or inhibit the activity or activation of CL-L2s protein, substances including antibodies to CL-L2s protein and the like (hereinafter, referred to as “CL-L2s related substance”). The CL-L2s related substances can be used neat, or after subjecting to several kinds of treatment such as dilution in water and the like, however, they can be used after blending in pharmaceutical products, quasi drugs and the like. In these cases, the amount of the substance to be blended may be determined ad libitum. When the substance is formulated for the systemic administration, 0.001-50% by weight, in particular, 0.01-10% by weight is permissible. When the amount is less than 0.001%, sufficient action of lacrimation may not be enabled. When the amount is greater than 50%, properties such as stability, flavor and the like of the composition itself may be deteriorated. [0160]
  • The route of administration can be optionally selected from the administration via mucosa, transdermal administration, intramuscular administration, subcutaneous administration, endorectal administration, topical ocular administration, and the like, in addition to oral administration and intravenous administration described above. [0161]
  • The CL-L2s related substance according to the present invention may be included in the formulation as a salt. Pharmaceutically acceptable salts include for example, salts with base such as inorganic base, organic base and the like; acid addition salts such as those of inorganic acid, organic acid, basic or acidic amino acid. Inorganic bases include for example, alkaline metal such as sodium, potassium and the like; alkaline earth metal such as calcium, magnesium and the like; aluminum, ammonium and the like. Organic bases include for example, primary amines such as ethanolamine and the like; secondary amines such as diethylamine, diethanolamine, dicyclohexylamine, N,N′-dibenzylethylenediamine and the like; tertiary amines such as trimethylamine, triethylamine, pyridine, picoline, triethanolamine and the like. Inorganic acids include for example, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Organic acids include for example, formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, benzoic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Basic amino acids include for example, arginine, lysine, ornithine and the like. Acidic amino acids include for example, aspartic acid, glutamic acid and the like. [0162]
  • Examples of dosage forms for use in oral administration include powdered formulations, granulated formulations, encapsulated formulations, pills, tablets, elixirs, suspensions, emulsions, syrups and the like, which may be selected ad libitum. In addition, such formulations may be modified, which may involve release control, stabilization, facilitation of disintegration, blocking of disintegration, enteric coating, facilitation of absorption and the like. Moreover, examples of dosage forms for the intraoral topical administration include chewable formulations, sublingual formulations, buccal formulations, lozenges, ointments, plasters, liquid formulations and the like, which may be selected ad libitum. Further, such formulations may be modified, which may involve release control, stabilization, facilitation of disintegration, blocking of disintegration, enteric coating, facilitation of absorption and the like. [0163]
  • Known drug delivery system (DDS) techniques may be applied to dosage forms as described above. DDS formulation referred to herein involves sustained release formulations, topically applied formulations (lozenges, buccal formulations, sublingual formulations), drug controlled release formulations, enteric coated formulations, formulations soluble in stomach and the like, which are formulations that are prepared so that most appropriate dosage form is accomplished taking into account of the administration route, bioavailability, adverse effect and the like. [0164]
  • Components for DDS essentially comprise a drug, a drug release module, a coating and a therapy program. In detail, the drug having a short half life is preferred, which permits rapid decline of the blood concentration particularly upon cessation of the release thereof. The coating is preferably nonreactive to the body tissue of the part to which the drug is administered. In addition, the therapy program is preferably configured so that the most optimal drug concentration is kept during the predetermined period. The drug release module substantially has a drug storage, a release control part, an energy source, and a release opening or a release surface. All of these fundamental components are not necessarily required, and thus addition, deletion or the like may be optionally carried out to select the best mode. [0165]
  • Examples of materials which can be used for DDS include polymers, cyclodextrin derivatives, lecithin and the like. The polymer may include insoluble polymers (silicone, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylcellulose, cellulose acetate and the like), water soluble polymers and hydroxyl gel-forming polymers (polyacrylamide, polyhydroxyethyl methacrylate cross-linked form, polyacryl cross-linked form, polyvinyl alcohol, polyethyleneoxide, water soluble cellulose derivatives, cross-linked poloxamer, chitin, chitosan and the like), slow dissolving polymers (ethyl cellulose, a partial ester of methylvinyl ether-maleic anhydride copolymer and the like), polymers soluble in stomach (hydroxylpropylmethyl cellulose, hydroxylpropyl cellulose, carmellose sodium, macrogol, polyvinylpyrrolidone, dimethylaminoethyl methacrylate-methyl methacrylate copolymer and the like), enteric polymers (hydroxylpropylmethyl cellulose phthalate, cellulose acetate phthalate, hydroxylpropylmethyl cellulose acetate succinate, carboxymethylethyl cellulose, acrylic acid polymers and the like), biodegradable polymers (heat coagulation or cross-linked albumin, cross-linked gelatin, collagen, fibrin, polycyanoacrylate, polyglycolic acid, polylactic acid, poly β-hydroxyacetic acid, polycaprolactone and the like), which can be selected ad libitum on the basis of the dosage form. [0166]
  • In particular, silicone, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, a partial ester of methylvinyl ether-maleic anhydride copolymer can be used for the control of drug release; cellulose acetate can be used as a material of a osmotic pressure pump; ethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose can be used as a material of a membrane of slow dissolving formulations; and polyacryl cross-linked form can be used as an attaching agent to mucosa. [0167]
  • Further, the formulation can be manufactured with adding solvent, excipient, coating agent, base, binding agent, lubricant, disintegrant, solubilizing agent, suspending agent, thickening agent, emulsifying agent, stabilizing agent, buffering agent, isotonizing agent, soothing agent, preservative agent, flavoring agent, fragrance agent, coloring agent and the like in compliance with its dosage form (known dosages form such as forms for oral administration, injection, suppository and the like). [0168]
  • Although specific examples are respectively illustrated below, these examples should not be construed as limiting the present invention. [0169]
  • [solvent] purified water, water for injection, saline, peanut oil, ethanol, glycerol; [0170]
  • [excipient] starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose, xylitol; [0171]
  • [coating agent] sucrose, gelatin, cellulose acetate phthalate and polymers as described above; [0172]
  • [base] vaseline, vegetable oil, macrogol, base for oil in water emulsion, base for water in oil emulsion; [0173]
  • [binding agent] natural polymer compounds such as starch and derivatives thereof, cellulose and derivatives thereof, gelatin, sodium alginate, gum tragacanth, gum arabic, and the like; synthetic polymers such as polyvinylpyrrolidone and the like; dextrin, hydroxylpropyl starch; [0174]
  • [lubricant] stearic acid and salts thereof, talc, waxes, wheat starch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol; [0175]
  • [disintegrant] starch and derivatives thereof, agar, gelatin powder, sodium bicarbonate, cellulose and derivatives thereof, carrnellose calcium, hydroxypropyl starch, carboxymethyl cellulose, and salts and derivatives thereof, poorly substituted hydroxypropyl cellulose; [0176]
  • [solubilizing agent] cyclodextrin, ethanol, propylene glycol, polyethylene glycol; [0177]
  • [suspending agent] gum arabic, gum tragacanth, sodium alginate, aluminum monostearate, citric acid, various surfactants; [0178]
  • [thickening agent] carmellose sodium, polyvinylpyrrolidone, methyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, gum tragacanth, gum arabic, sodium alginate; [0179]
  • [emulsifying agent] gum arabic, cholesterol, gum tragacanth, methyl cellulose, various surfactants, lecithin; [0180]
  • [stabilizing agent] sodium bisulfite, ascorbic acid, tocopherol, chelating agent, inert gas, reducing agent; [0181]
  • [buffering agent] sodium hydrogenphosphate, sodium acetate, boric acid; [0182]
  • [isotonizing agent] sodium chloride, glucose; [0183]
  • [soothing agent] procaine hydrochloride, lidocaine, benzyl alcohol; [0184]
  • [preservative agent] benzoic acid and salts thereof, p-hydroxybenzoic esters, chlorobutanol, inverted soap, benzyl alcohol, phenol, thimerosal; [0185]
  • [flavoring agent] sucrose, saccharin, glycyrrhiza extract, sorbitol, xylitol, glycerol; [0186]
  • [fragrance agent] orange peel tincture, rose oil; [0187]
  • [coloring agent] water soluble edible dye, lake dye. [0188]
    • EXAMPLES
  • Novel collectin according to the present invention is described in more detail by the following non-limiting illustrative examples. However, the present invention should not be construed to be limited by the examples. [0189]
  • Specifically, search on EST database (Example 1); screening of a novel human collectin from a cDNA library derived from human liver by PCR and sequencing of the base sequence (Example 2); screening of a novel human collectin a cap site cDNA library derived from human kidney by PCR and sequencing of the base sequence (Example 3); homology search of the novel collectin (Example 4); obtaining the novel mouse collectin cDNA (Example 5); analysis of distribution of expression of the novel human collectin in human tissues (Example 6); genetic analysis of the novel human collectin (Example 7); analysis of distribution of expression of the novel collectin CL-L2-1 and CL-L2-2 in human tissues (Example 8); construction of an expression vector pcDNA3.1/Myc-His(+)-CL-L2-1,2 of the novel collectin (Example 9); production of a cell strain that is stably expressing the novel collectin (Example 10); analysis of sugar specificity of the novel collectin (Example 11) are described below. [0190]
  • Example 1 [0191]
  • Search on EST Database [0192]
  • FIGS. 2 and 3 illustrate homology of amino acid residues of known collecting, i.e., human MBP, human SP-A, human SP-D and collectin CL-L1 derived from human liver, which was successfully isolated by the present inventor recently (see, Japanese patent unexamined publication No. Hei 11-206377), having a common structure as depicted in FIG. 1. In the Figure, portions of amino acid residues that are recognized as being homologous were boxed. An amino acid sequence of CRD (carbohydrate chain recognition domain) which is responsible for a lectin activity (SEQ ID NO: 14) of CL-L1 in this Figure was used to search on EST (Expressed Sequence Tags) database. [0193]
  • As a result, several data were obtained containing a highly homologous amino acid sequence. The amino acid sequences of thus resultant data were searched on GenBank/EST database, and determined whether they were either of known or unknown substance. Consequently, one datum (H30455, derived from thymus) that exhibits high homology but contains an unknown base sequence could be obtained. Using base sequence of the EST clone thus obtained, search of EST database was again conducted to give nine data (accession numbers: AA558494, derived from germ cells; AA582499, derived from kidney; AI420986, derived from prostate gland; AA742449, derived from germ cells; AA954657, derived from kidney; AA908360, derived from ovary; AI264145, derived from kidney; AA089855, derived from heart; AA456055, derived from melanocyte, pregnant uterus, fetal heart) that were found to include an identical base sequence. All of these were clones demonstrating a part of a base sequence of an identical novel collectin. [0194]
    • Example 2
  • Screening from a cDNA Library Derived from Human Liver by PCR and Sequencing of the Base Sequence [0195]
  • A consensus sequence (SEQ ID NO: 15) was produced in view of base sequences of 10 clones described above. Then, two primers toward the upstream direction: CAP1 (5′-agattttattgtatagcttgg-3′ (SEQ ID NO: 16)) and CAP2 (5′-ctgggtaataattacataatg-3′ (SEQ ID NO: 17) on the basis of a consensus sequence obtained in Example 1, and primes pertinent to a part of a region of a vector of the cDNA library derived from human liver: λTriplEx-F1 (5′-aagctccgagatctggacgag-3′ (SEQ ID NO: 18)) and λTriplEx-F2 (5′-ctcgggaagcgcgccattgtg-3′ (SEQ ID NO: 19)) were synthesized with 392A DNA/RNA synthesizer manufactured by PE Applied Biosystems Inc., and the screening by PCR was conducted as described below in order to clone a 5′ upstream region of a novel human collectin cDNA (see FIG. 4). [0196]
  • First PCR was conducted using a cDNA library derived from human (Clontech Co.,) as a template of the screening by PCR. Reaction mixture contained LA PCR Buffer II (Mg[0197] 2+ free), 2.5 mM MgCl2, each 1 μL of 200 μM dATP, dCTP, dGTP and dTTP (any of which is manufactured by Takara Shuzo Co., Ltd.), a cDNA library derived from human liver (Clontech Co.,), 0.5 μM λTriplEx-F1 primer and 0.5 μM CAP1 primer in a total volume of 50 μL. PCR was performed with a program of 35 cycles of heat denaturation at 95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation reaction at 72° C. for 90 seconds; and in addition, heat denaturation at 95° C. for 5 min prior to the repeated reaction and final elongation reaction at 72° C. for 5 min. After completing the first PCR, second PCR was conducted. The product of the first PCR was used as a template at 1 μL, and primers employed were λTriplEx-F2 primer and CAP2 primer. The reaction was performed with a similar reaction constitution and program to the first PCR except that the cycle number was 25 cycles. The PCR hereinabove was performed with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc. Thus resulting PCR product was verified on an agarose gel electrophoresis, and excised from the gel followed by freezing at −80° C. for 10 min. After centrifuging at 15000 rpm for 10 min, the product was purified by ethanol precipitation of the supernatant.
  • The purified DNA fragment was incorporated into pT7Blue Vector manufactured by Novagen CO., and the vector was transformed into competent cells, XL1-Blue cells. The transformant was cultured in a LB medium (100 μg/mL ampicillin) followed by extraction of the plasmid with an alkaline SDS method to sequence the base sequence thereof with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by Applied Biosystems Inc. Primers employed were M13 Universal Primer (5′-cgacgttgtaaaacgacggccagt-3′ (SEQ ID NO: 20)) and M13 Reverse Primer (5′-caggaaacagctatgac-3′ (SEQ ID NO: 21)), both of which were synthesized similarly to CAP1 primer. The base sequence accordingly obtained was revealed to be a base sequence having 575 bases longer than the CAP2 primer starting from 3′-end to N-terminal end thereof (a region corresponding to: the amino acid position of 68-271 of CL-L2-1 ORF shown in FIG. 4; or the amino acid position of 42-245 of CL-L2-2 ORF). However, slight difference was found from the base sequence of 5′-end region of the consensus sequence of EST obtained in Example 1. [0198]
    • Example 3
  • Screening of a Novel Human Collectin from a Cap Site cDNA Library Derived from Human Kidney by PCR and Sequencing of the Base Sequence [0199]
  • In order to clone 5′-end region containing a transcription initiation site in addition to the base sequence obtained in Example 2, screening by PCR was conducted using a cap site cDNA as follows through synthesizing two primers toward the upstream direction: CAP3 (5′-ggtcctatgtcaccggaatc-3′ (SEQ ID NO: 22)), CAP4 (5′-ttccatgacgacccacactgc-3′ (SEQ ID NO: 23)) with 392A DNA/RNA synthesizer manufactured by PE Applied Biosystems Inc., on the basis of the base sequence obtained in Example 2 (FIG. 4). [0200]
  • First PCR was performed with Cap Site cDNA, Human Kidney manufactured by NIPPON GENE Co., Ltd. using attached 1RC2 primer (5′-caaggtacgccacagcgtatg-3′ (SEQ ID NO: 24)) and CAP3 primer. Reaction mixture contained LA PCR Buffer II (Mg[0201] 2+ free), 2.5 mM MgCl2, each 1 μL of 200 μM dATP, dCTP, dGTP and dTTP (any of which is manufactured by Takara Shuzo Co., Ltd.), a Cap Site cDNA Human kidney, 0.5 μM 1RC2 primer (both of which were manufactured by NIPPON GENE Co., Ltd.), and 0.5 μM CAP3 primer in a total volume of 50 μL. PCR was performed with a program of 35 cycles of heat denaturation at 95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation reaction at 72° C. for 60 seconds; and in addition, heat denaturation at 95° C. for 5 min prior to the repeated reaction and final elongation reaction at 72° C. for 10 min. After completing the first PCR, second PCR was conducted. The product of the first PCR was used as a template at 1 μL, and primers employed were attached 2RC2 primer (5′-gtacgccacagcgtatgatgc-3′ (SEQ ID NO: 25)) and CAP4 primer. The reaction was performed with a similar reaction constitution and program to the first PCR except that the cycle number was 25 cycles. The PCR hereinabove was performed with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc. Thus resulting PCR product was verified on an agarose gel electrophoresis, and excised from the gel followed by freezing at −80° C. for 10 min. After centrifuging at 15000 rpm for 10 min, the product was purified by ethanol precipitation of the supernatant.
  • The purified DNA fragment was incorporated into pT7Blue Vector manufactured by Novagen CO., and the vector was transformed into competent cells, XLI-Blue cells. The transformant was cultured in a LB medium (100 μg/mL ampicillin) followed by extraction of the plasmid with an alkaline SDS method to sequence the base sequence thereof with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by Applied Biosystems Inc. Primers employed were M13 Universal Primer (5′-cgacgttgtaaaacgacggccagt-3′ (SEQ ID NO: 20)) and M13 Reverse Primer (5′-caggaaacagctatgac-3′ (SEQ ID NO: 21)). Two base sequences accordingly obtained were revealed to be: a base sequence having 492 bases longer than the base sequence obtained in Example 2 to its N-terminal direction (SEQ ID NO: 1); and a base sequence having 274 bases longer than the base sequence obtained in Example 2 to its N-terminal direction (SEQ ID NO: 3). [0202]
  • Consequently, two cDNAs in connection with CL-L2 were obtained hereby including: cDNA having an ORF (open reading frame) of 813 bases (SEQ ID NO: 1) and [0203] encoding 271 amino acids set out in SEQ ID NO: 2 (CL-L2-1); and cDNA having an ORF (open reading frame) of 735 bases (SEQ ID NO: 3) and encoding 245 amino acids set out in SEQ ID NO: 4 (CL-L2-2).
    • Example 4
  • Homology Search [0204]
  • Next, homology search was conducted for DNA and amino acid on GenBank database. As a result, the obtained amino acid sequence was demonstrated to be a trhat of a novel protein, which is distinct from any of collectins that have been found so far. [0205]
  • Amino acid sequences of three kinds of collectins reported so far (MBP, SP-A and SP-D) and collectin CL-L1 derived from human liver, which was successfully isolated by the present inventor recently (see, Japanese patent unexamined publication No. Hei 11-206377) were compared with the amino acid sequence of the collectin structural part of the novel collectin according to the present invention. The results are illustrated in FIG. 5 and FIG. 6. Similarly to FIGS. 2 and 3, portions of amino acid residues that are recognized as being homologous were boxed. According to this alignment, it was demonstrated that the resultant novel protein has homology to known collectin proteins, and that it belongs to a collectin family. [0206]
  • In addition, a mutant (SEQ ID NO: 6) encoded by the position of 141-731 of the base sequence set out in SEQ ID NO: 5 having deletion of amino acids of 18-65 in the amino acid sequence set out in SEQ ID NO: 4; a mutant (SEQ ID NO: 8) encoded by the position of 141-803 of the base sequence set out in SEQ ID NO: 7 having deletion of amino acids of 18-41 in the amino acid sequence set out in SEQ ID NO: 4; and a mutant (SEQ ID NO: 10) encoded by the position of 141-803 of the base sequence set out in SEQ ID NO: 9 having deletion of amino acids of 42-65 in the amino acid sequence set out in SEQ ID NO: 4 were obtained. [0207]
    • Example 5
  • Obtaining cDNA of Mouse Novel Mouse Collectin [0208]
  • In a similar manner to that for hCL-L2, mCL-L2 gene could be obtained through the screening of a mouse liver cDNA library.. The resulting cDNA clone of mCL-L2 was confirmed to have an ORF (open reading frame) of 813 bases (SEQ ID NO: 12), and encode amino acids of 271 amino acids set out in SEQ ID NO: 13. [0209]
    • Example 6
  • Analysis of Distribution of Expression of the Novel Collectin in Human Tissues [0210]
  • In order to examine the expression of the novel collectin in various tissues, analysis was performed by RT-PCR. RT-PCR was performed using two primers which are capable of amplifying a cDNA sequence spanning from neck region to carbohydrate recognition domain of the novel collectin: RTF1 (5′-agattccggtgacataggacc-3′ (SEQ ID NO: 26)), RTR1 (5′-tggtctgggctctgtccctgc-3′ (SEQ ID NO: 27)), and two primers which are capable of amplifying a part of β-actin gene for use in comparison of the amount of expressed novel collectin in each of the tissues: human β-actin sense primer (5′-caagagatggccacggctgct-3′ (SEQ ID NO: 28)), human β-actin antisense primer (5″-tccttctgcatcctgtcggca-3′ (SEQ ID NO: 29)). All of these primers were synthesized in a similar manner to CAP1 primer to conduct RT-PCR. [0211]
  • RT-PCR was carried out using RNA LA PCR Kit (AMV) Ver.1.1 (TAKARA Syuzo, Co.) with each RNA derived from several human tissues ((1) brain, (2) heart, (3) kidney, (4) liver, (5) lung, (6) trachea, (7) bone marrow, (8) colon, (9) small intestine, (10) spleen, (11) stomach, (12) thymus, (13) mammary gland, (14) prostate gland, (15) skeletal muscle, (16) testis, (17) uterus, (18) cerebellum, (19) fetal brain, (20) fetal liver, (21) spinal cord, (22) placenta, (23) adrenal gland, (24) pancreas, (25) salivary gland, and (26) thyroid) as a template. First, a reverse transcription reaction was conducted in the following reaction mixture. [0212]
  • The reaction mixture contained 5 mM MgCl[0213] 2, 1×RNA PCR Buffer, 1 mM dNTP Mixture, 1 U/μl RNase inhibitor and 2 μg of RNA, and total volume of the mixture was adjusted to give 40 μl with RNase free distilled water. At the same time, a reaction mixture without reverse transcriptase was also prepared for a negative control. The reaction mixture as described above was placed in 0.2 ml tube, and subjected to a reverse transcription reaction with GeneAmp PCR System9700 manufactured by PE Applied Biosystems Inc. through 1 cycle of: 30 minutes at 42° C., 5 minutes at 99° C., and 5 minutes at 5° C. Thus resulting reverse transcription reaction product was subsequently used at 10 μL for LA PCR in the following reaction mixture with respectively 28 cycles. and 35 cycles. Thereto were added 2.5 mM MgCl2, 1×LA PCR Buffer (Mg2+ free), 2U TaKaRa LA Taq, 0.2 μM RTF1 primer and 0.2 μM RTR1 primer, and the mixture was adjusted to give total volume of 50 μL with sterilized distilled water. PCR was performed with a program of 28 or 35 cycles of heat denaturation at 95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation reaction at 72° C. for 60 seconds; and in addition, heat denaturation at 95° C. for 5 min prior to the repeated reaction and final elongation reaction at 72° C. for 10 min. The reaction product was separated on 1.5% agarose gel electrophoresis, followed by staining with ethidium bromide solution (0.1 μg/mL), verification of the electrophoretic pattern with transilluminator, and identification of the expressing tissue. In order to compare the expressed amount in each of the tissues, RT-PCR was performed to amplify a part of β-actin with each of the tissues, and the correction of the amount of RNA was conducted. The RT-PCR was performed similarly to the above procedure with reverse transcription reaction, PCR, and the estimation was executed. The results are illustrated in FIG. 7, which demonstrate the expression of the novel collectin according to the present invention for PCR performed with 28 cycles in kidney (lane 3) intensively, and also in liver (lane 4), small intestine (lane 9), thymus (lane 12), fetal liver (lane 20), spinal cord (lane 21), adrenal gland (lane 23) and pancreas (lane 24). Further, with respect to PCR performed with 35 cycles, ubiquitous expression of the novel collectin could be verified in all tissues tested, although varying intensity of expression was observed.
    • Example 7
  • Genetic Analysis of the Novel Collectin [0214]
  • On the basis of sequence of DNAs of the obtained novel collectin (hCL-L2-1 (SEQ ID NO: 1) and mCL-L2 (SEQ ID NO: 12)), a phylogenetic tree was produced through conducting the analysis for the purpose of clarifying genetic position among the known collectins. [0215]
  • Collectins targeted for the analysis were proteins in various collecitn families illustrated in FIG. 8 (in the Figure, CL-L1 and CL-P1 were successfully isolated by the present inventors). Multiple alignment was produced with clustalw method using a region containing a lectin domain on the basis of data which were obtained by searching each amino acid sequences from GenBank database. A phylogenetic tree was produced with Phylip version 3.57 c package program using N-J process (neighbor-joining process) based on thus resulting multiple alignment. [0216]
  • Consequently, it was assumed that SP-D, bovine CL-43, and bovine conglutinin formed one cluster, whilst MBP and SP-A respectively form separate clusters, however, the novel collectin of the present invention did not belong in any of these clusters but belonged in the same cluster with CL-L1. Accordingly, it was speculated that the novel collectin of the present invention is a homologue of CL-L1. [0217]
    • Example 8
  • Analysis of Distribution of Expression of Novel Collectin (CL-L2-1 and CL-L2-2) in Human Tissues [0218]
  • Analysis with RT-PCR technique was conducted in order to examine expression of CL-L2-1 (SEQ ID NO: 1) and CL-L2-2 (SEQ ID NO: 3) in various tissues. RT PCR was conducted using primers which are capable of amplifying the entire translated region of CL-L2-1 as obtained (RTF2 (5′-atgagggggaatctggccctggtg-3′ (SEQ ID NO: 30)), RTR2 (5′-catgttctccttgtcaaactcac-3′ (SEQ ID NO: 31))); primers which are capable of amplifying the entire translated region of CL-L2-2 as obtained (RTF3 (5′-atgtggtgggtgcctccgagtc-3′ (SEQ ID NO: 32)), RTR2 (SEQ ID NO: 31)); and two human β-actin primers used in Example 6, which are capable of amplifying a part of β-actin gene for use in comparison of the amount of expressed novel collectin in each of the tissues. All of these primers were synthesized in a similar manner to CAP1 primer, and RT-PCR was performed similarly to Example 6. [0219]
  • The results are illustrated in FIG. 9, which demonstrate intensive expression of CL-L2-1 (SEQ ID NO: 1) according to the present invention for PCR performed with 28 cycles in kidney (lane 3), liver (lane 4), small intestine (lane 9), thymus (lane 12), fetal liver (lane 20), spinal cord (lane 21), adrenal gland (lane 23), pancreas (lane 24). Further, with respect to PCR performed with 35 cycles, ubiquitous expression of CL-L2-1 (SEQ ID NO: 1) could be verified in all tissues tested, although varying intensity of expression was observed. Moreover, it was found that CL-L2-2 (SEQ ID NO: 3) was intensively expressed in kidney (lane 3) for PCR performed with 28 cycles, whilst for PCR performed with 35 cycles was expressed in kidney (lane 3) as well as in prostate gland (lane 14), testis (lane 16), spinal cord (lane 21), placenta (lane 22). [0220]
  • Additionally, several amplified fragments were found in RT-PCR products of CL-L2-1 and CL-L2-2 as shown in FIG. 9. These bands were excised from the gel, and purified by a similar process to that in Example 2, i.e., by freezing at −80° C. for 10 min, centrifuging at 15000 rpm for 10 min followed by ethanol precipitation of the supernatant. Purified DNA fragment was incorporated into pT7Blue Vector manufactured by Novagen Co., and thus resulting vector was transformed into competent cells, XL1-Blue cells. The transformant was cultured in a LB medium (100 μg/ml ampicillin), and then a plasmid was extracted by an alkali SDS method. Base sequence was determined with BigDye Terminator Cycle Sequencing FS Ready Reaction kit and ABI PRISM 377 sequencer manufactured by PE Applied Biosystems Inc. Primers employed were M13 Universal Primer (5′-cgacgttgtaaaacgacggccagt-3′ (SEQ ID NO: 20)) and M13 Reverse Primer (5′-caggaaacagctatgac-3′ (SEQ ID NO: 21)). [0221]
  • Thus resulting base sequence was the same as CL-L2-2v1, CL-L2-2v2 and CL-L2-2v3 which are variants of CL-L2-2 (SEQ ID NO: 3) obtained in Example 2. Furhter, three proteins were present as CL-L2-1 set out in SEQ ID NO: 2, which were derived from alternative splicing of mRNA. Those are referred to as CL-L2-1v1 (SEQ ID NO: 36, 37), CL-L2-1v2 (SEQ ID NO: 38, 39) and CL-L2-1v3 (SEQ ID NO: 40, 41). CL-L2-1v1 has deletion of the amino acid position of 44-91 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 394-537 of CL-L2-1 set out in SEQ ID NO:1), whose amino acid sequence is encoded by the position of 265-933 of a base sequence set out in SEQ ID NO: 36 (SEQ ID NO: 59). CL-L2-1v2 has deletion of the amino acid position of 44-67 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 394-465 of CL-L2-1 set out in SEQ ID NO:1), whose amino acid sequence is encoded by the position of 265-1005 of a base sequence set out in SEQ ID NO: 38 (SEQ ID NO: 60). CL-L2-1v3 has deletion of the amino acid position of 68-91 of CL-L2-1 set out in SEQ ID NO: 2 (deletion of the base position of 466-537 of CL-L2-1 set out in SEQ ID NO: 1), whose amino acid sequence is encoded by the position of 265-1005 of a base sequence set out in SEQ ID NO: 40 (SEQ ID NO: 61). Moreover, mCL-L2-2 gene can be obtained in a similar process to that in Example 4. [0222]
    • Example 9
  • Construction of Expression Vector pcDNA3.1/Myc-His(+)A-CL-L2-1,2 of Novel Collectin [0223]
  • A translated region of CL-L2-1 (SEQ ID NO: 1) was amplified using CL-L2-1F primer (5′-gggaagcttcgatcaggatgagggggaatctggcctggtg-3′ (SEQ ID NO: 33)) and CL-L2-1R primer (5′-gggctcgagcatgttctccttgtcaaactcac-3′ (SEQ ID NO: 34)) by PCR (Takara Thermal Cycler MP manufactured by Takara Shuzo Co., Ltd.) with a cDNA library derived from human kidney as a template. Further, a translated region of the novel collectin (SEQ ID NO: 3) was amplified using CL-L2-2F primer (5′-gggaagcttccagcacaatgtggtgggtgcctccgagtc-3′ (SEQ ID NO: 35)) and CL-L2-1R primer (SEQ ID NO: 34) by PCR with a cDNA library derived from human kidney as a template. Thus resulting CL-L2-1 cDNA was ligated to pT7Blue T-Vector (manufactured by Novagen Co.) and was transformed into [0224] Escherichia coli XLI-Blue. A plasmid containing CL-L2-1 cDNA was purified from the resulting clone. Following the confirmation of the base sequence of the resulting plasmid with a sequencer, the plasmid with no error was digested with restriction enzymes Hind III and Xho I, and ligated to pcDNA3.1/Myc-His(+)A vector (manufactured by Invitrogen Co,.) that had been digested with the same enzymes and purified. After the ligated plasmid was transformed into Escherichia coli, XLI-Blue, the resulting clone was cultured. The plasmid was then purified to give an expression vector pcDNA3.1/Myc-His(+)A-CL-L2-1,2. At the same instant, expression vectors were similarly produced for variants of CL-L2-1 and CL-L2-2 (SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40).
    • Example 10
  • Production of Cell Strain which is Stably Expressing Novel Collectin [0225]
  • Transient expression was conducted through cotransfection of the expression vector pcDNA3.1/Myc-His(+)A-CL-L2-1 obtained in Example 9 and pEGFP-F vector (manufactured by Clontech Co.,) to CHO cells using LIPOFECTAMINE 2000 (LF2000) Reagent (manufactured by GIBCO BRL Co.). A 0.5 ml solution of LF2000 Reagent (LF2000 Reagent 30 μl, Nutrient Mixture F-12 Ham (Ham's F-12 medium, (manufactured by Sigma Co.))) was first prepared, and incubated at room temperature for 5 minutes. Then, 0.5 ml of a vector solution (pcDNA3.1/Myc-His(+)A-CL-L2-1,2: 7.5 μg, pEGFP-F vector 2.5 μg, Ham's F-12 medium) was admixed therewith, followed by incubation for 20 minutes. Thereafter, the solution was added to CHO cells that had been cultured to a high density in a 25 cm[0226] 2 flask including 5 ml of Ham's F-12 medium containing 5% FCS. After incubating at 37° C. for 4 hours in the presence of 5% CO2, the medium was replaced with a flesh medium, followed by subsequent incubation at 37° C. for 20 hours in the presence of 5% CO2. Next, the medium was replaced with Ham's F-12 medium containing 5% FCS, 0.4 mg/ml Geneticin, (manufactured by GIBCO BRL Co.), and 10 days culture was subsequently conducted. In this process, replacement of the medium was once carried out.
  • Through this selection by a drug for 10 days, only the transformed cells could survive and proliferated, however to the contrary, cells that were not transformed were dead. In order to obtain highly expressing cells from the resulting transformed cells, sorting was performed by a cell sorter (manufactured by Becton Dickinson Co.) with fluorescence of GFP as a marker. After washing the transformed cells in the 25 cm[0227] 2 flask with 5 ml PBS(−) twice, the cells were stripped off with 0.3 ml of 0.02% EDTA solution (manufactured by Nakarai Tesc KK). The cells were suspended in 10 ml PBS (−), and thereafter centrifuged at 200×g for 7 minutes at 4° C. to remove the supernatant. The remaining cells were suspended in 0.5 ml of 2% FCS/PBS (−) to give a sorting sample. After the sample was passed through a 5 ml tube equipped with a cell strainer cap (manufactured by Becton Dickinson Co.), it was applied to a cell sorter. CHO cells without subjecting to the transformation, which had been similarly treated, were used as control cells. Accordingly, a sample was selected, which exhibited fluorescence intensity of 10 times or greater than the control sample. These cells were dispensed into 96-well cell culture plates, of which wells respectively contained 100 μl Ham's F-12 medium (containing 5% FCS, 0.4 mg/ml Geneticin), to charge a single cell per well. After the cells were cultured at 37° C. in the presence of 5% CO2 for one week, each 100 μl of a culture medium was further added thereto followed by the additional culture for one week. A clone proliferated by the drug selection with Geneticin was divided into two parts, which were subjected to passages on 12-well and 24-well cell culture plates. Upon the passage, clones were excluded, which were derived from proliferation in a well where two or more cells existed per well, and the cells were plated at a cell number ratio of 9:1 for the 12-well and 24-well cell culture plates. The cells were cultured at 37° C. in the presence of 5% CO2 until the cells in the 12-well plate reach to high density. Then, 200 μl of the culture supernatant was dot blotted on an Immobilon-P membrane (manufactured by Millipore Co., Ltd.) using Bio-Dot Microfiltration Apparatus (manufactured by BIO-RAD Co., Ltd.), and the membrane was incubated in a solution of anti-myc antibody (manufactured by Invitorogen Co.,)×5000 diluted in 0.05% Tween 20/TBS buffer (manufactured by Takara Shuzo Co., Ltd.) at room temperature for 1 hour. Thereafter, the membrane was washed with 100 ml of 0.05% Tween 20/TBS buffer at room temperature for 20 min.×3 followed by further incubation in a solution of anti-IgG-HRP (manufactured by Chemicon Co., Ltd.)×5000 diluted in 0.05% Tween 20/TBS buffer at room temperature for 1 hour. Thereafter, the membrane was washed with 100 ml of 0.05% Tween 20/TBS buffer at room temperature for 20 minutes×3 followed by detection using TMB Membrane Peroxidase substrate system (manufactured by Funakoshi KK). After confirming the clones with intense color development, cells in respectively corresponding wells of the 24-well plate were identified as a stably expressing cell strain (CHO/CL-L2-1).
    • Example 11
  • Analysis of Sugar Binding Specificity of Novel Collectin [0228]
  • One litter of the culture supernatant of the stably expressing cell strain of the novel collectin (CHO/CL-L2-1) produced in Example 10 was concentrated to 50 ml using VIVAPORE10 (manufactured by Funakoshi KK), thereafter was added 200 μl of Ni-NTA agarose (manufactured by Quiagen Co., Ltd.) thereto. The novel collectin was bound to Ni-NTA agarose by incubation of the mixture with shaking at 4° C. overnight. Ni-NTA agarose was packed in Poly-Prep Chromatography Columns (manufactured by BIO-RAD Co., Ltd.) followed by washes with 5 ml of 50 mM NaH[0229] 2PO4, 300 mM NaCl, 20 mM imidazole, 0.05% Tween20 (pH8.0) three times, and by elution with 200 μl of 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 0.05% Tween20 (pH8.0) five times to purify the novel collectin. Purified novel collectin was quantitatively determined, and used for the analysis of sugar specificity.
  • Specifically, 1 μg of the purified novel collectin was dot blotted on an Immobilon-P membrane (manufactured by Millipore Co., Ltd.) to give spots in a number corresponding to types of sugar chain probes to be examined for binding specificity, using Bio-Dot Microfiltration Apparatus (manufactured by BIO-RAD Co., Ltd.). Each dot was thereafter cut to yield 1 cm square, and was immersed in Block Ace (manufactured by Dai Nippon Pharmaceutical Co., Ltd.), and was incubated at room temperature for 1 hour. Next, the membrane was washed with 0.05% Tween20, 5 mM CaCl[0230] 2, TBS buffer three times, and was incubated in each sugar chain probe solution (see, FIG. 10) diluted with 5 mM CaCl2, TBS buffer to give 1 μg/ml at room temperature for 1 hour. Thereafter, the membrane was again washed with 0.05% Tween20, 5mM CaCl2, TBS buffer three times. Then the membrane was incubated in a solution of Streptabvidin-biotinylated HRP (manufactured by Amersham Co.)×1000 diluted in 0.05% Tween20, 5 mM CaCl2, TBS buffer at room temperature for 30 min. followed by washes with 0.05% Tween20, 5mM CaCl2, TBS buffer three times and by detection using TMB Membrane Peroxidase substrate system (manufactured by Funakoshi KK). As is shown in FIG. 10, the novel collectin was active in binding to mannose, fucose, N-acetylgalactosamine, N-acetylneuraminic acid, mannose-6-phosphoric acid.
  • Because CL-L2s protein of the present invention has a collectin structure, it is believed to be a substance that exerts characteristic effects to those structures. Therefore, it can be utilized in the elucidation of mechanisms of fundamental immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and in the development of reagents and drugs for the same. [0231]
  • 1 61 1 1341 DNA Homo Sapiens CDS (265)..(1077) 1 cgcggccgcg tcgacggacg gtggacgcag cgcagacagg aagctccccg agataacgct 60 gcggccgggc ggcctgattt gctgggctgt ctgatggccc gggccgaggc ttctccctgc 120 gcctgggact gcggccgcct ctctaaatag cagccatgag gcgcctgggg gcagtgtcct 180 cgcggccgcg tcgaccgacg gccgcagtcg acgccccgtt cgcctagcgc gtgctcagga 240 gttggtgtcc tgcctgcgct cagg atg agg ggg aat ctg gcc ctg gtg ggc 291 Met Arg Gly Asn Leu Ala Leu Val Gly 1 5 gtt cta atc agc ctg gcc ttc ctg tca ctg ctg cca tct gga cat cct 339 Val Leu Ile Ser Leu Ala Phe Leu Ser Leu Leu Pro Ser Gly His Pro 10 15 20 25 cag ccg gct ggc gat gac gcc tgc tct gtg cag atc ctc gtc cct ggc 387 Gln Pro Ala Gly Asp Asp Ala Cys Ser Val Gln Ile Leu Val Pro Gly 30 35 40 ctc aaa ggg gat gcg gga gag aag gga gac aaa ggc gcc ccc gga cgg 435 Leu Lys Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg 45 50 55 cct gga aga gtc ggc ccc acg gga gaa aaa gga gac atg ggg gac aaa 483 Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Asp Met Gly Asp Lys 60 65 70 gga cag aaa ggc agt gtg ggt cgt cat gga aaa att ggt ccc att ggc 531 Gly Gln Lys Gly Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile Gly 75 80 85 tct aaa ggt gag aaa gga gat tcc ggt gac ata gga ccc cct ggt cct 579 Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro 90 95 100 105 aat gga gaa cca ggc ctc cca tgt gag tgc agc cag ctg cgc aag gcc 627 Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala 110 115 120 atc ggg gag atg gac aac cag gtc tct cag ctg acc agc gag ctc aag 675 Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys 125 130 135 ttc atc aag aat gct gtc gcc ggt gtg cgc gag acg gag agc aag atc 723 Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile 140 145 150 tac ctg ctg gtg aag gag gag aag cgc tac gcg gac gcc cag ctg tcc 771 Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser 155 160 165 tgc cag ggc cgc ggg ggc acg ctg agc atg ccc aag gac gag gct gcc 819 Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala 170 175 180 185 aat ggc ctg atg gcc gca tac ctg gcg caa gcc ggc ctg gcc cgt gtc 867 Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val 190 195 200 ttc atc ggc atc aac gac ctg gag aag gag ggc gcc ttc gtg tac tct 915 Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser 205 210 215 gac cac tcc ccc atg cgg acc ttc aac aag tgg cgc agc ggt gag ccc 963 Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro 220 225 230 aac aat gcc tac gac gag gag gac tgc gtg gag atg gtg gcc tcg ggc 1011 Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly 235 240 245 ggc tgg aac gac gtg gcc tgc cac acc acc atg tac ttc atg tgt gag 1059 Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu 250 255 260 265 ttt gac aag gag aac atg tgagcctcag gctggggctg cccattgggg 1107 Phe Asp Lys Glu Asn Met 270 gccccacatg tccctgcagg gttggcaggg acagagccca gaccatggtg ccagccaggg 1167 agctgtccct ctgtgaaggg tggaggctca ctgagtagag ggctgttgtc taaactgaga 1227 aaatggccta tgcttaagag gaaaatgaaa gtgttcctgg ggtgctgtct ctgaagaagc 1287 agagtttcat tacctgtatt gtagccccaa tgtcattatg taattattac ccag 1341 2 271 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO1. 2 Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Asp Ala Gly Glu 35 40 45 Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro Gly Arg Val Gly Pro Thr 50 55 60 Gly Glu Lys Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly 65 70 75 80 Arg His Gly Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp 85 90 95 Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 100 105 110 Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln 115 120 125 Val Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala 130 135 140 Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu 145 150 155 160 Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr 165 170 175 Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr 180 185 190 Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu 195 200 205 Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr 210 215 220 Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu 225 230 235 240 Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys 245 250 255 His Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 260 265 270 3 1139 DNA Homo Sapiens CDS (141)..(875) 3 cagaagtttt ggtgaaagtg gctttggccc tgactttgtg gtagcgtgtg tgggtttgtg 60 agtggaacct tcagctttag gttggaaacg gtggctgtgg agagctggac ttttggctgt 120 ggaggtcacg tccctgccca atg tgg tgg gtg cct ccg agt ccc tac ggt tgt 173 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys 1 5 10 ctt ccc tgc gcc ctg cca ggg gat gcg gga gag aag gga gac aaa ggc 221 Leu Pro Cys Ala Leu Pro Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly 15 20 25 gcc ccc gga cgg cct gga aga gtc ggc ccc acg gga gaa aaa gga gac 269 Ala Pro Gly Arg Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Asp 30 35 40 atg ggg gac aaa gga cag aaa ggc agt gtg ggt cgt cat gga aaa att 317 Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly Arg His Gly Lys Ile 45 50 55 ggt ccc att ggc tct aaa ggt gag aaa gga gat tcc ggt gac ata gga 365 Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly 60 65 70 75 ccc cct ggt cct aat gga gaa cca ggc ctc cca tgt gag tgc agc cag 413 Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln 80 85 90 ctg cgc aag gcc atc ggg gag atg gac aac cag gtc tct cag ctg acc 461 Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr 95 100 105 agc gag ctc aag ttc atc aag aat gct gtc gcc ggt gtg cgc gag acg 509 Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr 110 115 120 gag agc aag atc tac ctg ctg gtg aag gag gag aag cgc tac gcg gac 557 Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp 125 130 135 gcc cag ctg tcc tgc cag ggc cgc ggg ggc acg ctg agc atg ccc aag 605 Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys 140 145 150 155 gac gag gct gcc aat ggc ctg atg gcc gca tac ctg gcg caa gcc ggc 653 Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly 160 165 170 ctg gcc cgt gtc ttc atc ggc atc aac gac ctg gag aag gag ggc gcc 701 Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala 175 180 185 ttc gtg tac tct gac cac tcc ccc atg cgg acc ttc aac aag tgg cgc 749 Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg 190 195 200 agc ggt gag ccc aac aat gcc tac gac gag gag gac tgc gtg gag atg 797 Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met 205 210 215 gtg gcc tcg ggc ggc tgg aac gac gtg gcc tgc cac acc acc atg tac 845 Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr 220 225 230 235 ttc atg tgt gag ttt gac aag gag aac atg tgagcctcag gctggggctg 895 Phe Met Cys Glu Phe Asp Lys Glu Asn Met 240 245 cccattgggg gccccacatg tccctgcagg gttggcaggg acagagccca gaccatggtg 955 ccagccaggg agctgtccct ctgtgaaggg tggaggctca ctgagtagag ggctgttgtc 1015 taaactgaga aaatggccta tgcttaagag gaaaatgaaa gtgttcctgg ggtgctgtct 1075 ctgaagaagc agagtttcat tacctgtatt gtagccccaa tgtcattatg taattattac 1135 ccag 1139 4 245 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO3. 4 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys Leu Pro Cys Ala Leu 1 5 10 15 Pro Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro 20 25 30 Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Asp Met Gly Asp Lys Gly 35 40 45 Gln Lys Gly Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile Gly Ser 50 55 60 Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn 65 70 75 80 Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile 85 90 95 Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys Phe 100 105 110 Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr 115 120 125 Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys 130 135 140 Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala Asn 145 150 155 160 Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val Phe 165 170 175 Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp 180 185 190 His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn 195 200 205 Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly Gly 210 215 220 Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu Phe 225 230 235 240 Asp Lys Glu Asn Met 245 5 995 DNA Homo Sapiens CDS (141)..(731) 5 cagaagtttt ggtgaaagtg gctttggccc tgactttgtg gtagcgtgtg tgggtttgtg 60 agtggaacct tcagctttag gttggaaacg gtggctgtgg agagctggac ttttggctgt 120 ggaggtcacg tccctgccca atg tgg tgg gtg cct ccg agt ccc tac ggt tgt 173 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys 1 5 10 ctt ccc tgc gcc ctg cca ggt gag aaa gga gat tcc ggt gac ata gga 221 Leu Pro Cys Ala Leu Pro Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly 15 20 25 ccc cct ggt cct aat gga gaa cca ggc ctc cca tgt gag tgc agc cag 269 Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln 30 35 40 ctg cgc aag gcc atc ggg gag atg gac aac cag gtc tct cag ctg acc 317 Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr 45 50 55 agc gag ctc aag ttc atc aag aat gct gtc gcc ggt gtg cgc gag acg 365 Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr 60 65 70 75 gag agc aag atc tac ctg ctg gtg aag gag gag aag cgc tac gcg gac 413 Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp 80 85 90 gcc cag ctg tcc tgc cag ggc cgc ggg ggc acg ctg agc atg ccc aag 461 Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys 95 100 105 gac gag gct gcc aat ggc ctg atg gcc gca tac ctg gcg caa gcc ggc 509 Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly 110 115 120 ctg gcc cgt gtc ttc atc ggc atc aac gac ctg gag aag gag ggc gcc 557 Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala 125 130 135 ttc gtg tac tct gac cac tcc ccc atg cgg acc ttc aac aag tgg cgc 605 Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg 140 145 150 155 agc ggt gag ccc aac aat gcc tac gac gag gag gac tgc gtg gag atg 653 Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met 160 165 170 gtg gcc tcg ggc ggc tgg aac gac gtg gcc tgc cac acc acc atg tac 701 Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr 175 180 185 ttc atg tgt gag ttt gac aag gag aac atg tgagcctcag gctggggctg 751 Phe Met Cys Glu Phe Asp Lys Glu Asn Met 190 195 cccattgggg gccccacatg tccctgcagg gttggcaggg acagagccca gaccatggtg 811 ccagccaggg agctgtccct ctgtgaaggg tggaggctca ctgagtagag ggctgttgtc 871 taaactgaga aaatggccta tgcttaagag gaaaatgaaa gtgttcctgg ggtgctgtct 931 ctgaagaagc agagtttcat tacctgtatt gtagccccaa tgtcattatg taattattac 991 ccag 995 6 197 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO5. 6 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys Leu Pro Cys Ala Leu 1 5 10 15 Pro Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn 20 25 30 Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile 35 40 45 Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys Phe 50 55 60 Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr 65 70 75 80 Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys 85 90 95 Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala Asn 100 105 110 Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val Phe 115 120 125 Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp 130 135 140 His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn 145 150 155 160 Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly Gly 165 170 175 Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu Phe 180 185 190 Asp Lys Glu Asn Met 195 7 1067 DNA Homo Sapiens CDS (141)..(803) 7 cagaagtttt ggtgaaagtg gctttggccc tgactttgtg gtagcgtgtg tgggtttgtg 60 agtggaacct tcagctttag gttggaaacg gtggctgtgg agagctggac ttttggctgt 120 ggaggtcacg tccctgccca atg tgg tgg gtg cct ccg agt ccc tac ggt tgt 173 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys 1 5 10 ctt ccc tgc gcc ctg cca gga gac atg ggg gac aaa gga cag aaa ggc 221 Leu Pro Cys Ala Leu Pro Gly Asp Met Gly Asp Lys Gly Gln Lys Gly 15 20 25 agt gtg ggt cgt cat gga aaa att ggt ccc att ggc tct aaa ggt gag 269 Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu 30 35 40 aaa gga gat tcc ggt gac ata gga ccc cct ggt cct aat gga gaa cca 317 Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro 45 50 55 ggc ctc cca tgt gag tgc agc cag ctg cgc aag gcc atc ggg gag atg 365 Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met 60 65 70 75 gac aac cag gtc tct cag ctg acc agc gag ctc aag ttc atc aag aat 413 Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn 80 85 90 gct gtc gcc ggt gtg cgc gag acg gag agc aag atc tac ctg ctg gtg 461 Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val 95 100 105 aag gag gag aag cgc tac gcg gac gcc cag ctg tcc tgc cag ggc cgc 509 Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg 110 115 120 ggg ggc acg ctg agc atg ccc aag gac gag gct gcc aat ggc ctg atg 557 Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met 125 130 135 gcc gca tac ctg gcg caa gcc ggc ctg gcc cgt gtc ttc atc ggc atc 605 Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile 140 145 150 155 aac gac ctg gag aag gag ggc gcc ttc gtg tac tct gac cac tcc ccc 653 Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro 160 165 170 atg cgg acc ttc aac aag tgg cgc agc ggt gag ccc aac aat gcc tac 701 Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr 175 180 185 gac gag gag gac tgc gtg gag atg gtg gcc tcg ggc ggc tgg aac gac 749 Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp 190 195 200 gtg gcc tgc cac acc acc atg tac ttc atg tgt gag ttt gac aag gag 797 Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu 205 210 215 aac atg tgagcctcag gctggggctg cccattgggg gccccacatg tccctgcagg 853 Asn Met 220 gttggcaggg acagagccca gaccatggtg ccagccaggg agctgtccct ctgtgaaggg 913 tggaggctca ctgagtagag ggctgttgtc taaactgaga aaatggccta tgcttaagag 973 gaaaatgaaa gtgttcctgg ggtgctgtct ctgaagaagc agagtttcat tacctgtatt 1033 gtagccccaa tgtcattatg taattattac ccag 1067 8 221 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO7. 8 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys Leu Pro Cys Ala Leu 1 5 10 15 Pro Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly Arg His 20 25 30 Gly Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly 35 40 45 Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu 50 55 60 Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val Ser 65 70 75 80 Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly Val 85 90 95 Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg 100 105 110 Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser 115 120 125 Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala 130 135 140 Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu Lys 145 150 155 160 Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe Asn 165 170 175 Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys 180 185 190 Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His Thr 195 200 205 Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 210 215 220 9 1067 DNA Homo Sapiens CDS (141)..(803) 9 cagaagtttt ggtgaaagtg gctttggccc tgactttgtg gtagcgtgtg tgggtttgtg 60 agtggaacct tcagctttag gttggaaacg gtggctgtgg agagctggac ttttggctgt 120 ggaggtcacg tccctgccca atg tgg tgg gtg cct ccg agt ccc tac ggt tgt 173 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys 1 5 10 ctt ccc tgc gcc ctg cca ggg gat gcg gga gag aag gga gac aaa ggc 221 Leu Pro Cys Ala Leu Pro Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly 15 20 25 gcc ccc gga cgg cct gga aga gtc ggc ccc acg gga gaa aaa ggt gag 269 Ala Pro Gly Arg Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Glu 30 35 40 aaa gga gat tcc ggt gac ata gga ccc cct ggt cct aat gga gaa cca 317 Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro 45 50 55 ggc ctc cca tgt gag tgc agc cag ctg cgc aag gcc atc ggg gag atg 365 Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met 60 65 70 75 gac aac cag gtc tct cag ctg acc agc gag ctc aag ttc atc aag aat 413 Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn 80 85 90 gct gtc gcc ggt gtg cgc gag acg gag agc aag atc tac ctg ctg gtg 461 Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val 95 100 105 aag gag gag aag cgc tac gcg gac gcc cag ctg tcc tgc cag ggc cgc 509 Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg 110 115 120 ggg ggc acg ctg agc atg ccc aag gac gag gct gcc aat ggc ctg atg 557 Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met 125 130 135 gcc gca tac ctg gcg caa gcc ggc ctg gcc cgt gtc ttc atc ggc atc 605 Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile 140 145 150 155 aac gac ctg gag aag gag ggc gcc ttc gtg tac tct gac cac tcc ccc 653 Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro 160 165 170 atg cgg acc ttc aac aag tgg cgc agc ggt gag ccc aac aat gcc tac 701 Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr 175 180 185 gac gag gag gac tgc gtg gag atg gtg gcc tcg ggc ggc tgg aac gac 749 Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp 190 195 200 gtg gcc tgc cac acc acc atg tac ttc atg tgt gag ttt gac aag gag 797 Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu 205 210 215 aac atg tgagcctcag gctggggctg cccattgggg gccccacatg tccctgcagg 853 Asn Met 220 gttggcaggg acagagccca gaccatggtg ccagccaggg agctgtccct ctgtgaaggg 913 tggaggctca ctgagtagag ggctgttgtc taaactgaga aaatggccta tgcttaagag 973 gaaaatgaaa gtgttcctgg ggtgctgtct ctgaagaagc agagtttcat tacctgtatt 1033 gtagccccaa tgtcattatg taattattac ccag 1067 10 221 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO9. 10 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys Leu Pro Cys Ala Leu 1 5 10 15 Pro Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro 20 25 30 Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Glu Lys Gly Asp Ser Gly 35 40 45 Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu 50 55 60 Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val Ser 65 70 75 80 Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly Val 85 90 95 Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg 100 105 110 Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser 115 120 125 Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala 130 135 140 Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu Lys 145 150 155 160 Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe Asn 165 170 175 Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys 180 185 190 Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His Thr 195 200 205 Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 210 215 220 11 45 PRT Homo Sapiens Deduced Amino Acid Sequence of a collagen-like domain of mutated Novel Collectin. 11 Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro Gly 1 5 10 15 Arg Val Gly Pro Thr Gly Glu Lys Gly Glu Lys Gly Asp Ser Gly Asp 20 25 30 Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 35 40 45 12 1522 DNA Mus musculus CDS (157)..(969) 12 cagcccagcg aatctctctg agctggactg cagctggtct ctgtaaataa cagccatgag 60 gctccttgga gcagtgtctc tgccagccgg tgactgtcag gacagctcct tcgcctagtg 120 tgcatccagg agtctagtgt cctgcctgca ctcatg atg atg agg gac ctg gct 174 Met Met Arg Asp Leu Ala 1 5 ctt gca ggc atg ctg att agc ctg gct ttc ctg tcc ctg ctg cca tct 222 Leu Ala Gly Met Leu Ile Ser Leu Ala Phe Leu Ser Leu Leu Pro Ser 10 15 20 gga tgt cct cag cag acc aca gag gac gcc tgc tct gtg cag att ctt 270 Gly Cys Pro Gln Gln Thr Thr Glu Asp Ala Cys Ser Val Gln Ile Leu 25 30 35 gtc ccc ggc ctc aaa ggg gat gca gga gaa aag gga gac aaa gga gcc 318 Val Pro Gly Leu Lys Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala 40 45 50 cca gga cgg cca gga aga gtc ggc cct aca gga gaa aaa gga gac atg 366 Pro Gly Arg Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Asp Met 55 60 65 70 ggg gac aaa gga cag aaa ggc act gtg ggc cgc cat gga aaa att ggt 414 Gly Asp Lys Gly Gln Lys Gly Thr Val Gly Arg His Gly Lys Ile Gly 75 80 85 ccc att ggc gca aaa ggt gaa aaa gga gat tct ggt gat atc gga ccc 462 Pro Ile Gly Ala Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro 90 95 100 cct ggc ccc agt gga gaa cct ggt att cca tgt gag tgc agt cag ctg 510 Pro Gly Pro Ser Gly Glu Pro Gly Ile Pro Cys Glu Cys Ser Gln Leu 105 110 115 agg aag gct att ggg gag atg gac aac cag gtc act caa ctg aca act 558 Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val Thr Gln Leu Thr Thr 120 125 130 gag cta aaa ttc ata aaa aat gct gtt gct ggc gtg cgc gag act gag 606 Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu 135 140 145 150 agc aag atc tac ctg ctg gtg aag gag gag aag cgg tac gca gat gcc 654 Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala 155 160 165 cag ctg tcc tgc caa gcc cga ggc ggc aca ctg agc atg ccc aaa gac 702 Gln Leu Ser Cys Gln Ala Arg Gly Gly Thr Leu Ser Met Pro Lys Asp 170 175 180 gag gca gcc aat ggc ctg atg gct tca tac ctg gca cag gct ggc ctg 750 Glu Ala Ala Asn Gly Leu Met Ala Ser Tyr Leu Ala Gln Ala Gly Leu 185 190 195 gcc cga gtc ttc atc ggt atc aat gac ctg gag aaa gaa ggt gct ttc 798 Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe 200 205 210 gtg tac tcg gac cgc tcc ccc atg cag acc ttc aac aag tgg cgc agt 846 Val Tyr Ser Asp Arg Ser Pro Met Gln Thr Phe Asn Lys Trp Arg Ser 215 220 225 230 gga gag ccc aac aac gcc tat gat gag gag gac tgt gtg gag atg gtg 894 Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val 235 240 245 gcc tca ggt ggc tgg aat gat gtg gcc tgc cac att acc atg tac ttc 942 Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His Ile Thr Met Tyr Phe 250 255 260 atg tgc gag ttt gac aaa gag aac ttg tgagagccga caggggagat 989 Met Cys Glu Phe Asp Lys Glu Asn Leu 265 270 ggccatctga acgccacctt ttatagacac cagcggccac aaactaaccc tgagcaccag 1049 tcgccatgtc tgcgggttcc tctctgcatg gaagtgccgg gcctcattga cagttggaag 1109 ggctgtttga accgtaggag gggagaaacc ttgcttccgg ggctgttctg aataggtggc 1169 gtattatcac ctttgtcaga cgttcattat ggctaaccac tcggaaggat gctgtgaagc 1229 tcttgtcttg gtccagcata gtaaattttg cagcagtcat caaactggct ataggggcag 1289 gagttgccgc ccaccctata aagtacacag agtgcccagg tggtgaccaa tgtttctata 1349 ggttatgcta tcacatagat tcctttcact attatccggg taggaagact gctgctctgc 1409 ttcacatcca tatttcagga aaacaaataa atcctctgat ttctgctaaa aaaaaaaaaa 1469 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 1522 13 271 PRT Mus musculus Deduced Amino Acid Sequence of Novel Collectin from Nucleotide Sequence set out in SEQ ID NO12. 13 Met Met Arg Asp Leu Ala Leu Ala Gly Met Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly Cys Pro Gln Gln Thr Thr Glu Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Asp Ala Gly Glu 35 40 45 Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro Gly Arg Val Gly Pro Thr 50 55 60 Gly Glu Lys Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Thr Val Gly 65 70 75 80 Arg His Gly Lys Ile Gly Pro Ile Gly Ala Lys Gly Glu Lys Gly Asp 85 90 95 Ser Gly Asp Ile Gly Pro Pro Gly Pro Ser Gly Glu Pro Gly Ile Pro 100 105 110 Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln 115 120 125 Val Thr Gln Leu Thr Thr Glu Leu Lys Phe Ile Lys Asn Ala Val Ala 130 135 140 Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu 145 150 155 160 Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Ala Arg Gly Gly Thr 165 170 175 Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ser Tyr 180 185 190 Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu 195 200 205 Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp Arg Ser Pro Met Gln Thr 210 215 220 Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu 225 230 235 240 Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys 245 250 255 His Ile Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Leu 260 265 270 14 159 PRT Homo sapiens Known CRD amino acid sequence of reported CL-L1 which was employed for searching EST data base. 14 Cys Asp Cys Gly Arg Tyr Arg Lys Phe Val Gly Gln Leu Asp Ile Ser 1 5 10 15 Ile Ala Arg Leu Lys Thr Ser Met Lys Phe Val Lys Asn Val Ile Ala 20 25 30 Gly Ile Arg Glu Thr Glu Glu Lys Phe Tyr Tyr Ile Val Gln Glu Glu 35 40 45 Lys Asn Tyr Arg Glu Ser Leu Thr His Cys Arg Ile Arg Gly Gly Met 50 55 60 Leu Ala Met Pro Lys Asp Glu Ala Ala Asn Thr Leu Ile Ala Asp Tyr 65 70 75 80 Val Ala Lys Ser Gly Phe Phe Arg Val Phe Ile Gly Val Asn Asp Leu 85 90 95 Glu Arg Glu Gly Gln Tyr Met Phe Thr Asp Asn Thr Pro Leu Gln Asn 100 105 110 Tyr Ser Asn Trp Asn Glu Gly Glu Pro Ser Asp Pro Tyr Gly His Glu 115 120 125 Asp Cys Val Glu Met Leu Ser Ser Gly Arg Trp Asn Asp Thr Glu Cys 130 135 140 His Leu Thr Met Tyr Phe Val Cys Glu Phe Ile Lys Lys Lys Lys 145 150 155 15 619 DNA Artificial Sequence Consensus nucleotide sequence of novel collectin derived from several nucleotide sequences obtained from EST data base. 15 ttctcagatg attcctgtgc atggcgaagt ttggaaactc tgagtgttgc ttttgagatc 60 agacatacag tgctatccat tggtgccctc gtggaggatg gaacttactt gttgactnnt 120 gccacaccac catgtacttc atgtgtgagt ttgacaagga gaacatgtga ccctcaggct 180 ggggctgccc attgggggcc ccacatgtcc ctgcagggtt ggcagggaca gagcccagac 240 catggtgcca gccagggagc tgtccctctg tgaagggtgg aggctcactg agtagagggc 300 tgttgtctaa actgagaaaa tggcctatgc ttaagaggaa aatgaaagtg ttcctggggt 360 gctgtctctg aagaagcaga gtttcattac ctgtattgta gccccaatgt cattatgtaa 420 ttattaccca gaattgctct tccataaagc ttgtgccttt gtccaagcta tacaataaaa 480 tctttaagta gtgcagtagt taagtccaaa aagtggcaat ggggtcttga aaaaaaaaaa 540 aaaaatttat aaaaaaaaaa gaactcactt tgaccaacac ttctgtaaat tacattacaa 600 tataggttcc ttcacacta 619 16 21 DNA Artificial Sequence Synthetic primer CAP1 for cloning 5′-upstream region of novel collectin. 16 agattttatt gtatagcttg g 21 17 21 DNA Artificial Sequence Synthetic primer CAP2 for cloning 5′-upstream region of novel collectin. 17 ctgggtaata attacataat g 21 18 21 DNA Artificial Sequence Synthetic primer TriplEx-F1 for cloning 5′-upstream region of novel collectin. 18 aagctccgag atctggacga g 21 19 21 DNA Artificial Sequence Synthetic primer TriplEx-F2 for cloning 5′-upstream region of novel collectin. 19 ctcgggaagc gcgccattgt g 21 20 24 DNA Artificial Sequence Synthetic M13 Universal primer for sequencing novel collectin. 20 cgacgttgta aaacgacggc cagt 24 21 17 DNA Artificial Sequence Synthetic M13 Reverse primer for sequencing novel collectin. 21 caggaaacag ctatgac 17 22 20 DNA Artificial Sequence Synthetic primer CAP3 for further cloning of 5′-upstream region of novel collectin. 22 ggtcctatgt caccggaatc 20 23 21 DNA Artificial Sequence Synthetic primer CAP4 for further cloning of 5′-upstream region of novel collectin. 23 ttccatgacg acccacactg c 21 24 21 DNA Artificial Sequence Synthetic primer 1RC2 for further cloning of 5′-upstream region of novel collectin. 24 caaggtacgc cacagcgtat g 21 25 21 DNA Artificial Sequence Synthetic primer 2RC2 for further cloning of 5′-upstream region of novel collectin. 25 gtacgccaca gcgtatgatg c 21 26 21 DNA Artificial Sequence Synthetic primer RTF1 for RT-PCR to determine tissue distribution of novel collectin. 26 agattccggt gacataggac c 21 27 21 DNA Artificial Sequence Synthetic primer RTR1 for RT-PCR to determine tissue distribution of novel collectin. 27 tggtctgggc tctgtccctg c 21 28 21 DNA Artificial Sequence Synthetic actin sense primer for RT-PCR to determine control level. 28 caagagatgg ccacggctgc t 21 29 21 DNA Artificial Sequence Synthetic actin anti-sense primer for RT-PCR to determine control level. 29 tccttctgca tcctgtcggc a 21 30 24 DNA Artificial Sequence Synthetic primer RTF2 for amplification of CL-L2-1. 30 atgaggggga atctggccct ggtg 24 31 23 DNA Artificial Sequence Synthetic primer RTR2 for amplification of CL-L2-1. 31 catgttctcc ttgtcaaact cac 23 32 22 DNA Artificial Sequence Synthetic primer RTF3 for amplification of CL-L2-2. 32 atgtggtggg tgcctccgag tc 22 33 41 DNA Artificial Sequence Synthetic primer CL-L2-1F for amplification of CL-L2-1. 33 gggaagcttc gatcaggatg agggggaatc tggccctggt g 41 34 32 DNA Artificial Sequence Synthetic primer CL-L2-1R for amplification of CL-L2-1. 34 gggctcgagc atgttctcct tgtcaaactc ac 32 35 46 DNA Artificial Sequence Synthetic primer CL-L2-2F for amplification of CL-L2-2. 35 gggaagcttc cagcacaatg tggtgggtgc ctccgagtcc ctggtg 46 36 1197 DNA Homo Sapiens CDS (265)..(933) 36 cgcggccgcg tcgacggacg gtggacgcag cgcagacagg aagctccccg agataacgct 60 gcggccgggc ggcctgattt gctgggctgt ctgatggccc gggccgaggc ttctccctgc 120 gcctgggact gcggccgcct ctctaaatag cagccatgag gcgcctgggg gcagtgtcct 180 cgcggccgcg tcgaccgacg gccgcagtcg acgccccgtt cgcctagcgc gtgctcagga 240 gttggtgtcc tgcctgcgct cagg atg agg ggg aat ctg gcc ctg gtg ggc 291 Met Arg Gly Asn Leu Ala Leu Val Gly 1 5 gtt cta atc agc ctg gcc ttc ctg tca ctg ctg cca tct gga cat cct 339 Val Leu Ile Ser Leu Ala Phe Leu Ser Leu Leu Pro Ser Gly His Pro 10 15 20 25 cag ccg gct ggc gat gac gcc tgc tct gtg cag atc ctc gtc cct ggc 387 Gln Pro Ala Gly Asp Asp Ala Cys Ser Val Gln Ile Leu Val Pro Gly 30 35 40 ctc aaa ggt gag aaa gga gat tcc ggt gac ata gga ccc cct ggt cct 435 Leu Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro 45 50 55 aat gga gaa cca ggc ctc cca tgt gag tgc agc cag ctg cgc aag gcc 483 Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys Ala 60 65 70 atc ggg gag atg gac aac cag gtc tct cag ctg acc agc gag ctc aag 531 Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu Lys 75 80 85 ttc atc aag aat gct gtc gcc ggt gtg cgc gag acg gag agc aag atc 579 Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys Ile 90 95 100 105 tac ctg ctg gtg aag gag gag aag cgc tac gcg gac gcc cag ctg tcc 627 Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser 110 115 120 tgc cag ggc cgc ggg ggc acg ctg agc atg ccc aag gac gag gct gcc 675 Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala 125 130 135 aat ggc ctg atg gcc gca tac ctg gcg caa gcc ggc ctg gcc cgt gtc 723 Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val 140 145 150 ttc atc ggc atc aac gac ctg gag aag gag ggc gcc ttc gtg tac tct 771 Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser 155 160 165 gac cac tcc ccc atg cgg acc ttc aac aag tgg cgc agc ggt gag ccc 819 Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro 170 175 180 185 aac aat gcc tac gac gag gag gac tgc gtg gag atg gtg gcc tcg ggc 867 Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly 190 195 200 ggc tgg aac gac gtg gcc tgc cac acc acc atg tac ttc atg tgt gag 915 Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys Glu 205 210 215 ttt gac aag gag aac atg tgagcctcag gctggggctg cccattgggg 963 Phe Asp Lys Glu Asn Met 220 gccccacatg tccctgcagg gttggcaggg acagagccca gaccatggtg ccagccaggg 1023 agctgtccct ctgtgaaggg tggaggctca ctgagtagag ggctgttgtc taaactgaga 1083 aaatggccta tgcttaagag gaaaatgaaa gtgttcctgg ggtgctgtct ctgaagaagc 1143 agagtttcat tacctgtatt gtagccccaa tgtcattatg taattattac ccag 1197 37 223 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin variant from Nucleotide Sequence set out in SEQ ID NO36. 37 Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Glu Lys Gly Asp 35 40 45 Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 50 55 60 Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln 65 70 75 80 Val Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala 85 90 95 Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu 100 105 110 Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr 115 120 125 Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr 130 135 140 Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu 145 150 155 160 Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr 165 170 175 Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu 180 185 190 Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys 195 200 205 His Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 210 215 220 38 1269 DNA Homo Sapiens CDS (265)..(1005) 38 cgcggccgcg tcgacggacg gtggacgcag cgcagacagg aagctccccg agataacgct 60 gcggccgggc ggcctgattt gctgggctgt ctgatggccc gggccgaggc ttctccctgc 120 gcctgggact gcggccgcct ctctaaatag cagccatgag gcgcctgggg gcagtgtcct 180 cgcggccgcg tcgaccgacg gccgcagtcg acgccccgtt cgcctagcgc gtgctcagga 240 gttggtgtcc tgcctgcgct cagg atg agg ggg aat ctg gcc ctg gtg ggc 291 Met Arg Gly Asn Leu Ala Leu Val Gly 1 5 gtt cta atc agc ctg gcc ttc ctg tca ctg ctg cca tct gga cat cct 339 Val Leu Ile Ser Leu Ala Phe Leu Ser Leu Leu Pro Ser Gly His Pro 10 15 20 25 cag ccg gct ggc gat gac gcc tgc tct gtg cag atc ctc gtc cct ggc 387 Gln Pro Ala Gly Asp Asp Ala Cys Ser Val Gln Ile Leu Val Pro Gly 30 35 40 ctc aaa gga gac atg ggg gac aaa gga cag aaa ggc agt gtg ggt cgt 435 Leu Lys Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly Arg 45 50 55 cat gga aaa att ggt ccc att ggc tct aaa ggt gag aaa gga gat tcc 483 His Gly Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp Ser 60 65 70 ggt gac ata gga ccc cct ggt cct aat gga gaa cca ggc ctc cca tgt 531 Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys 75 80 85 gag tgc agc cag ctg cgc aag gcc atc ggg gag atg gac aac cag gtc 579 Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val 90 95 100 105 tct cag ctg acc agc gag ctc aag ttc atc aag aat gct gtc gcc ggt 627 Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly 110 115 120 gtg cgc gag acg gag agc aag atc tac ctg ctg gtg aag gag gag aag 675 Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys 125 130 135 cgc tac gcg gac gcc cag ctg tcc tgc cag ggc cgc ggg ggc acg ctg 723 Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu 140 145 150 agc atg ccc aag gac gag gct gcc aat ggc ctg atg gcc gca tac ctg 771 Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu 155 160 165 gcg caa gcc ggc ctg gcc cgt gtc ttc atc ggc atc aac gac ctg gag 819 Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu 170 175 180 185 aag gag ggc gcc ttc gtg tac tct gac cac tcc ccc atg cgg acc ttc 867 Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe 190 195 200 aac aag tgg cgc agc ggt gag ccc aac aat gcc tac gac gag gag gac 915 Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp 205 210 215 tgc gtg gag atg gtg gcc tcg ggc ggc tgg aac gac gtg gcc tgc cac 963 Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His 220 225 230 acc acc atg tac ttc atg tgt gag ttt gac aag gag aac atg 1005 Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 235 240 245 tgagcctcag gctggggctg cccattgggg gccccacatg tccctgcagg gttggcaggg 1065 acagagccca gaccatggtg ccagccaggg agctgtccct ctgtgaaggg tggaggctca 1125 ctgagtagag ggctgttgtc taaactgaga aaatggccta tgcttaagag gaaaatgaaa 1185 gtgttcctgg ggtgctgtct ctgaagaagc agagtttcat tacctgtatt gtagccccaa 1245 tgtcattatg taattattac ccag 1269 39 247 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin variant from Nucleotide Sequence set out in SEQ ID NO38. 39 Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Asp Met Gly Asp 35 40 45 Lys Gly Gln Lys Gly Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile 50 55 60 Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly 65 70 75 80 Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys 85 90 95 Ala Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu 100 105 110 Lys Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys 115 120 125 Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu 130 135 140 Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala 145 150 155 160 Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg 165 170 175 Val Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr 180 185 190 Ser Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu 195 200 205 Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser 210 215 220 Gly Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys 225 230 235 240 Glu Phe Asp Lys Glu Asn Met 245 40 1269 DNA Homo Sapiens CDS (265)..(1005) 40 cgcggccgcg tcgacggacg gtggacgcag cgcagacagg aagctccccg agataacgct 60 gcggccgggc ggcctgattt gctgggctgt ctgatggccc gggccgaggc ttctccctgc 120 gcctgggact gcggccgcct ctctaaatag cagccatgag gcgcctgggg gcagtgtcct 180 cgcggccgcg tcgaccgacg gccgcagtcg acgccccgtt cgcctagcgc gtgctcagga 240 gttggtgtcc tgcctgcgct cagg atg agg ggg aat ctg gcc ctg gtg ggc 291 Met Arg Gly Asn Leu Ala Leu Val Gly 1 5 gtt cta atc agc ctg gcc ttc ctg tca ctg ctg cca tct gga cat cct 339 Val Leu Ile Ser Leu Ala Phe Leu Ser Leu Leu Pro Ser Gly His Pro 10 15 20 25 cag ccg gct ggc gat gac gcc tgc tct gtg cag atc ctc gtc cct ggc 387 Gln Pro Ala Gly Asp Asp Ala Cys Ser Val Gln Ile Leu Val Pro Gly 30 35 40 ctc aaa ggg gat gcg gga gag aag gga gac aaa ggc gcc ccc gga cgg 435 Leu Lys Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg 45 50 55 cct gga aga gtc ggc ccc acg gga gaa aaa ggt gag aaa gga gat tcc 483 Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Glu Lys Gly Asp Ser 60 65 70 ggt gac ata gga ccc cct ggt cct aat gga gaa cca ggc ctc cca tgt 531 Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys 75 80 85 gag tgc agc cag ctg cgc aag gcc atc ggg gag atg gac aac cag gtc 579 Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val 90 95 100 105 tct cag ctg acc agc gag ctc aag ttc atc aag aat gct gtc gcc ggt 627 Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala Gly 110 115 120 gtg cgc gag acg gag agc aag atc tac ctg ctg gtg aag gag gag aag 675 Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu Lys 125 130 135 cgc tac gcg gac gcc cag ctg tcc tgc cag ggc cgc ggg ggc acg ctg 723 Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr Leu 140 145 150 agc atg ccc aag gac gag gct gcc aat ggc ctg atg gcc gca tac ctg 771 Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr Leu 155 160 165 gcg caa gcc ggc ctg gcc cgt gtc ttc atc ggc atc aac gac ctg gag 819 Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu Glu 170 175 180 185 aag gag ggc gcc ttc gtg tac tct gac cac tcc ccc atg cgg acc ttc 867 Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr Phe 190 195 200 aac aag tgg cgc agc ggt gag ccc aac aat gcc tac gac gag gag gac 915 Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu Asp 205 210 215 tgc gtg gag atg gtg gcc tcg ggc ggc tgg aac gac gtg gcc tgc cac 963 Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys His 220 225 230 acc acc atg tac ttc atg tgt gag ttt gac aag gag aac atg 1005 Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 235 240 245 tgagcctcag gctggggctg cccattgggg gccccacatg tccctgcagg gttggcaggg 1065 acagagccca gaccatggtg ccagccaggg agctgtccct ctgtgaaggg tggaggctca 1125 ctgagtagag ggctgttgtc taaactgaga aaatggccta tgcttaagag gaaaatgaaa 1185 gtgttcctgg ggtgctgtct ctgaagaagc agagtttcat tacctgtatt gtagccccaa 1245 tgtcattatg taattattac ccag 1269 41 247 PRT Homo Sapiens Deduced Amino Acid Sequence of Novel Collectin variant from Nucleotide Sequence set out in SEQ ID NO40. 41 Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Asp Ala Gly Glu 35 40 45 Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro Gly Arg Val Gly Pro Thr 50 55 60 Gly Glu Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly 65 70 75 80 Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu Cys Ser Gln Leu Arg Lys 85 90 95 Ala Ile Gly Glu Met Asp Asn Gln Val Ser Gln Leu Thr Ser Glu Leu 100 105 110 Lys Phe Ile Lys Asn Ala Val Ala Gly Val Arg Glu Thr Glu Ser Lys 115 120 125 Ile Tyr Leu Leu Val Lys Glu Glu Lys Arg Tyr Ala Asp Ala Gln Leu 130 135 140 Ser Cys Gln Gly Arg Gly Gly Thr Leu Ser Met Pro Lys Asp Glu Ala 145 150 155 160 Ala Asn Gly Leu Met Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg 165 170 175 Val Phe Ile Gly Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr 180 185 190 Ser Asp His Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu 195 200 205 Pro Asn Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser 210 215 220 Gly Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys 225 230 235 240 Glu Phe Asp Lys Glu Asn Met 245 42 48 PRT Homo Sapiens Deduced Amino Acid Sequence of a collagen-like domain of mutated Novel Collectin. 42 Gly Leu Lys Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly 1 5 10 15 Arg His Gly Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp 20 25 30 Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 35 40 45 43 24 PRT Homo Sapiens Deduced Amino Acid Sequence of a collagen-like domain of mutated Novel Collectin. 43 Gly Leu Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly 1 5 10 15 Pro Asn Gly Glu Pro Gly Leu Pro 20 44 48 PRT Homo Sapiens Deduced Amino Acid Sequence of a collagen-like domain of mutated Novel Collectin. 44 Gly Leu Lys Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly 1 5 10 15 Arg Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Glu Lys Gly Asp 20 25 30 Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 35 40 45 45 813 DNA Homo Sapiens 45 atgaggggga atctggccct ggtgggcgtt ctaatcagcc tggccttcct gtcactgctg 60 ccatctggac atcctcagcc ggctggcgat gacgcctgct ctgtgcagat cctcgtccct 120 ggcctcaaag gggatgcggg agagaaggga gacaaaggcg cccccggacg gcctggaaga 180 gtcggcccca cgggagaaaa aggagacatg ggggacaaag gacagaaagg cagtgtgggt 240 cgtcatggaa aaattggtcc cattggctct aaaggtgaga aaggagattc cggtgacata 300 ggaccccctg gtcctaatgg agaaccaggc ctcccatgtg agtgcagcca gctgcgcaag 360 gccatcgggg agatggacaa ccaggtctct cagctgacca gcgagctcaa gttcatcaag 420 aatgctgtcg ccggtgtgcg cgagacggag agcaagatct acctgctggt gaaggaggag 480 aagcgctacg cggacgccca gctgtcctgc cagggccgcg ggggcacgct gagcatgccc 540 aaggacgagg ctgccaatgg cctgatggcc gcatacctgg cgcaagccgg cctggcccgt 600 gtcttcatcg gcatcaacga cctggagaag gagggcgcct tcgtgtactc tgaccactcc 660 cccatgcgga ccttcaacaa gtggcgcagc ggtgagccca acaatgccta cgacgaggag 720 gactgcgtgg agatggtggc ctcgggcggc tggaacgacg tggcctgcca caccaccatg 780 tacttcatgt gtgagtttga caaggagaac atg 813 46 735 DNA Homo Sapiens 46 atgtggtggg tgcctccgag tccctacggt tgtcttccct gcgccctgcc aggggatgcg 60 ggagagaagg gagacaaagg cgcccccgga cggcctggaa gagtcggccc cacgggagaa 120 aaaggagaca tgggggacaa aggacagaaa ggcagtgtgg gtcgtcatgg aaaaattggt 180 cccattggct ctaaaggtga gaaaggagat tccggtgaca taggaccccc tggtcctaat 240 ggagaaccag gcctcccatg tgagtgcagc cagctgcgca aggccatcgg ggagatggac 300 aaccaggtct ctcagctgac cagcgagctc aagttcatca agaatgctgt cgccggtgtg 360 cgcgagacgg agagcaagat ctacctgctg gtgaaggagg agaagcgcta cgcggacgcc 420 cagctgtcct gccagggccg cgggggcacg ctgagcatgc ccaaggacga ggctgccaat 480 ggcctgatgg ccgcatacct ggcgcaagcc ggcctggccc gtgtcttcat cggcatcaac 540 gacctggaga aggagggcgc cttcgtgtac tctgaccact cccccatgcg gaccttcaac 600 aagtggcgca gcggtgagcc caacaatgcc tacgacgagg aggactgcgt ggagatggtg 660 gcctcgggcg gctggaacga cgtggcctgc cacaccacca tgtacttcat gtgtgagttt 720 gacaaggaga acatg 735 47 159 PRT Homo Sapiens 47 Cys Glu Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln 1 5 10 15 Val Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala 20 25 30 Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu Glu 35 40 45 Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly Gly Thr 50 55 60 Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met Ala Ala Tyr 65 70 75 80 Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly Ile Asn Asp Leu 85 90 95 Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His Ser Pro Met Arg Thr 100 105 110 Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn Asn Ala Tyr Asp Glu Glu 115 120 125 Asp Cys Val Glu Met Val Ala Ser Gly Gly Trp Asn Asp Val Ala Cys 130 135 140 His Thr Thr Met Tyr Phe Met Cys Glu Phe Asp Lys Glu Asn Met 145 150 155 48 477 DNA Homo Sapiens 48 tgtgagtgca gccagctgcg caaggccatc ggggagatgg acaaccaggt ctctcagctg 60 accagcgagc tcaagttcat caagaatgct gtcgccggtg tgcgcgagac ggagagcaag 120 atctacctgc tggtgaagga ggagaagcgc tacgcggacg cccagctgtc ctgccagggc 180 cgcgggggca cgctgagcat gcccaaggac gaggctgcca atggcctgat ggccgcatac 240 ctggcgcaag ccggcctggc ccgtgtcttc atcggcatca acgacctgga gaaggagggc 300 gccttcgtgt actctgacca ctcccccatg cggaccttca acaagtggcg cagcggtgag 360 cccaacaatg cctacgacga ggaggactgc gtggagatgg tggcctcggg cggctggaac 420 gacgtggcct gccacaccac catgtacttc atgtgtgagt ttgacaagga gaacatg 477 49 72 PRT Homo Sapiens 49 Gly Leu Lys Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly 1 5 10 15 Arg Pro Gly Arg Val Gly Pro Thr Gly Glu Lys Gly Asp Met Gly Asp 20 25 30 Lys Gly Gln Lys Gly Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile 35 40 45 Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly 50 55 60 Pro Asn Gly Glu Pro Gly Leu Pro 65 70 50 69 PRT Homo Sapiens 50 Gly Asp Ala Gly Glu Lys Gly Asp Lys Gly Ala Pro Gly Arg Pro Gly 1 5 10 15 Arg Val Gly Pro Thr Gly Glu Lys Gly Asp Met Gly Asp Lys Gly Gln 20 25 30 Lys Gly Ser Val Gly Arg His Gly Lys Ile Gly Pro Ile Gly Ser Lys 35 40 45 Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly 50 55 60 Glu Pro Gly Leu Pro 65 51 21 PRT Homo Sapiens 51 Gly Glu Lys Gly Asp Ser Gly Asp Ile Gly Pro Pro Gly Pro Asn Gly 1 5 10 15 Glu Pro Gly Leu Pro 20 52 45 PRT Homo Sapiens 52 Gly Asp Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly Arg His Gly 1 5 10 15 Lys Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp 20 25 30 Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro 35 40 45 53 40 PRT Homo Sapiens 53 Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala Phe 1 5 10 15 Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp Asp Ala 20 25 30 Cys Ser Val Gln Ile Leu Val Pro 35 40 54 17 PRT Homo Sapiens 54 Met Trp Trp Val Pro Pro Ser Pro Tyr Gly Cys Leu Pro Cys Ala Leu 1 5 10 15 Pro 55 591 DNA Homo Sapiens 55 atgtggtggg tgcctccgag tccctacggt tgtcttccct gcgccctgcc aggtgagaaa 60 ggagattccg gtgacatagg accccctggt cctaatggag aaccaggcct cccatgtgag 120 tgcagccagc tgcgcaaggc catcggggag atggacaacc aggtctctca gctgaccagc 180 gagctcaagt tcatcaagaa tgctgtcgcc ggtgtgcgcg agacggagag caagatctac 240 ctgctggtga aggaggagaa gcgctacgcg gacgcccagc tgtcctgcca gggccgcggg 300 ggcacgctga gcatgcccaa ggacgaggct gccaatggcc tgatggccgc atacctggcg 360 caagccggcc tggcccgtgt cttcatcggc atcaacgacc tggagaagga gggcgccttc 420 gtgtactctg accactcccc catgcggacc ttcaacaagt ggcgcagcgg tgagcccaac 480 aatgcctacg acgaggagga ctgcgtggag atggtggcct cgggcggctg gaacgacgtg 540 gcctgccaca ccaccatgta cttcatgtgt gagtttgaca aggagaacat g 591 56 663 DNA Homo Sapiens 56 atgtggtggg tgcctccgag tccctacggt tgtcttccct gcgccctgcc aggagacatg 60 ggggacaaag gacagaaagg cagtgtgggt cgtcatggaa aaattggtcc cattggctct 120 aaaggtgaga aaggagattc cggtgacata ggaccccctg gtcctaatgg agaaccaggc 180 ctcccatgtg agtgcagcca gctgcgcaag gccatcgggg agatggacaa ccaggtctct 240 cagctgacca gcgagctcaa gttcatcaag aatgctgtcg ccggtgtgcg cgagacggag 300 agcaagatct acctgctggt gaaggaggag aagcgctacg cggacgccca gctgtcctgc 360 cagggccgcg ggggcacgct gagcatgccc aaggacgagg ctgccaatgg cctgatggcc 420 gcatacctgg cgcaagccgg cctggcccgt gtcttcatcg gcatcaacga cctggagaag 480 gagggcgcct tcgtgtactc tgaccactcc cccatgcgga ccttcaacaa gtggcgcagc 540 ggtgagccca acaatgccta cgacgaggag gactgcgtgg agatggtggc ctcgggcggc 600 tggaacgacg tggcctgcca caccaccatg tacttcatgt gtgagtttga caaggagaac 660 atg 663 57 663 DNA Homo Sapiens 57 atgtggtggg tgcctccgag tccctacggt tgtcttccct gcgccctgcc aggggatgcg 60 ggagagaagg gagacaaagg cgcccccgga cggcctggaa gagtcggccc cacgggagaa 120 aaaggtgaga aaggagattc cggtgacata ggaccccctg gtcctaatgg agaaccaggc 180 ctcccatgtg agtgcagcca gctgcgcaag gccatcgggg agatggacaa ccaggtctct 240 cagctgacca gcgagctcaa gttcatcaag aatgctgtcg ccggtgtgcg cgagacggag 300 agcaagatct acctgctggt gaaggaggag aagcgctacg cggacgccca gctgtcctgc 360 cagggccgcg ggggcacgct gagcatgccc aaggacgagg ctgccaatgg cctgatggcc 420 gcatacctgg cgcaagccgg cctggcccgt gtcttcatcg gcatcaacga cctggagaag 480 gagggcgcct tcgtgtactc tgaccactcc cccatgcgga ccttcaacaa gtggcgcagc 540 ggtgagccca acaatgccta cgacgaggag gactgcgtgg agatggtggc ctcgggcggc 600 tggaacgacg tggcctgcca caccaccatg tacttcatgt gtgagtttga caaggagaac 660 atg 663 58 813 DNA Mus musculus 58 atgatgaggg acctggctct tgcaggcatg ctgattagcc tggctttcct gtccctgctg 60 ccatctggat gtcctcagca gaccacagag gacgcctgct ctgtgcagat tcttgtcccc 120 ggcctcaaag gggatgcagg agaaaaggga gacaaaggag ccccaggacg gccaggaaga 180 gtcggcccta caggagaaaa aggagacatg ggggacaaag gacagaaagg cactgtgggc 240 cgccatggaa aaattggtcc cattggcgca aaaggtgaaa aaggagattc tggtgatatc 300 ggaccccctg gccccagtgg agaacctggt attccatgtg agtgcagtca gctgaggaag 360 gctattgggg agatggacaa ccaggtcact caactgacaa ctgagctaaa attcataaaa 420 aatgctgttg ctggcgtgcg cgagactgag agcaagatct acctgctggt gaaggaggag 480 aagcggtacg cagatgccca gctgtcctgc caagcccgag gcggcacact gagcatgccc 540 aaagacgagg cagccaatgg cctgatggct tcatacctgg cacaggctgg cctggcccga 600 gtcttcatcg gtatcaatga cctggagaaa gaaggtgctt tcgtgtactc ggaccgctcc 660 cccatgcaga ccttcaacaa gtggcgcagt ggagagccca acaacgccta tgatgaggag 720 gactgtgtgg agatggtggc ctcaggtggc tggaatgatg tggcctgcca cattaccatg 780 tacttcatgt gcgagtttga caaagagaac ttg 813 59 669 DNA Homo Sapiens 59 atgaggggga atctggccct ggtgggcgtt ctaatcagcc tggccttcct gtcactgctg 60 ccatctggac atcctcagcc ggctggcgat gacgcctgct ctgtgcagat cctcgtccct 120 ggcctcaaag gtgagaaagg agattccggt gacataggac cccctggtcc taatggagaa 180 ccaggcctcc catgtgagtg cagccagctg cgcaaggcca tcggggagat ggacaaccag 240 gtctctcagc tgaccagcga gctcaagttc atcaagaatg ctgtcgccgg tgtgcgcgag 300 acggagagca agatctacct gctggtgaag gaggagaagc gctacgcgga cgcccagctg 360 tcctgccagg gccgcggggg cacgctgagc atgcccaagg acgaggctgc caatggcctg 420 atggccgcat acctggcgca agccggcctg gcccgtgtct tcatcggcat caacgacctg 480 gagaaggagg gcgccttcgt gtactctgac cactccccca tgcggacctt caacaagtgg 540 cgcagcggtg agcccaacaa tgcctacgac gaggaggact gcgtggagat ggtggcctcg 600 ggcggctgga acgacgtggc ctgccacacc accatgtact tcatgtgtga gtttgacaag 660 gagaacatg 669 60 741 DNA Homo Sapiens 60 atgaggggga atctggccct ggtgggcgtt ctaatcagcc tggccttcct gtcactgctg 60 ccatctggac atcctcagcc ggctggcgat gacgcctgct ctgtgcagat cctcgtccct 120 ggcctcaaag gagacatggg ggacaaagga cagaaaggca gtgtgggtcg tcatggaaaa 180 attggtccca ttggctctaa aggtgagaaa ggagattccg gtgacatagg accccctggt 240 cctaatggag aaccaggcct cccatgtgag tgcagccagc tgcgcaaggc catcggggag 300 atggacaacc aggtctctca gctgaccagc gagctcaagt tcatcaagaa tgctgtcgcc 360 ggtgtgcgcg agacggagag caagatctac ctgctggtga aggaggagaa gcgctacgcg 420 gacgcccagc tgtcctgcca gggccgcggg ggcacgctga gcatgcccaa ggacgaggct 480 gccaatggcc tgatggccgc atacctggcg caagccggcc tggcccgtgt cttcatcggc 540 atcaacgacc tggagaagga gggcgccttc gtgtactctg accactcccc catgcggacc 600 ttcaacaagt ggcgcagcgg tgagcccaac aatgcctacg acgaggagga ctgcgtggag 660 atggtggcct cgggcggctg gaacgacgtg gcctgccaca ccaccatgta cttcatgtgt 720 gagtttgaca aggagaacat g 741 61 741 DNA Homo Sapiens 61 atgaggggga atctggccct ggtgggcgtt ctaatcagcc tggccttcct gtcactgctg 60 ccatctggac atcctcagcc ggctggcgat gacgcctgct ctgtgcagat cctcgtccct 120 ggcctcaaag gggatgcggg agagaaggga gacaaaggcg cccccggacg gcctggaaga 180 gtcggcccca cgggagaaaa aggtgagaaa ggagattccg gtgacatagg accccctggt 240 cctaatggag aaccaggcct cccatgtgag tgcagccagc tgcgcaaggc catcggggag 300 atggacaacc aggtctctca gctgaccagc gagctcaagt tcatcaagaa tgctgtcgcc 360 ggtgtgcgcg agacggagag caagatctac ctgctggtga aggaggagaa gcgctacgcg 420 gacgcccagc tgtcctgcca gggccgcggg ggcacgctga gcatgcccaa ggacgaggct 480 gccaatggcc tgatggccgc atacctggcg caagccggcc tggcccgtgt cttcatcggc 540 atcaacgacc tggagaagga gggcgccttc gtgtactctg accactcccc catgcggacc 600 ttcaacaagt ggcgcagcgg tgagcccaac aatgcctacg acgaggagga ctgcgtggag 660 atggtggcct cgggcggctg gaacgacgtg gcctgccaca ccaccatgta cttcatgtgt 720 gagtttgaca aggagaacat g 741

Claims (40)

What is claimed is:
1. A protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 2, or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2, or a derivative or a fragment thereof.
2. A base sequence set out in SEQ ID NO: 45 (corresponding to a base sequence set out in the base position 265-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 2.
3. A protein comprising an amino acid sequence consisting of 245 amino acids set out in the amino acid position 1-245 of SEQ ID NO: 4, or a protein consisting of an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4, or a derivative or a fragment thereof.
4. A base sequence set out in SEQ ID NO: 46 (corresponding to a base sequence set out in the base position 141-875 of SEQ ID NO: 3), a base sequence encoding an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-245 of SEQ ID NO: 4.
5. A protein comprising an amino acid sequence consisting of 159 amino acids set out in SEQ ID NO: 47 (corresponding to an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2), or a protein consisting of an amino acid sequence set out in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2, or a derivative or a fragment thereof.
6. A base sequence set out in SEQ ID NO: 48 (corresponding to a base sequence set out in the base position 601-1077 of SEQ ID NO: 1), a base sequence encoding an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 113-271 of SEQ ID NO: 2.
7. The protein further comprising a sequence (Gly-Xaa-Yaa)n, wherein n represents an integer of from 1 to 50, Xaa and Yaa represent an amino acid residue, which may be the same or different, at an N-terminal of the protein according to claim 5.
8. The protein according to claim 7 wherein said (Gly-Xaa-Yaa)n is selected from the group of sequences consisting of:
Gly-Leu-Lys-Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg—Val-Gly-Pro-Thr-Gly-Glu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly—Arg-His-Gly-Lys-Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 49 (corresponding to the amino acid position 41-112 of SEQ ID NO: 2)),
Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg-Val-Gly-Pro—Thr-Gly-Glu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly—Lys-Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 50 (corresponding to the amino acid position 44-112 of SEQ ID NO: 2, or the amino acid position 18-86 of SEQ ID NO: 4)) Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 51 (corresponding to the amino acid position 92-112 of SEQ ID NO: 2 or the amino acid position 66-86 of SEQ ID NO: 4)),
Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly-Lys-Ile-Gly-Pro—Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 52 (corresponding to the amino acid position 68-112 of SEQ ID NO: 2 or the amino acid position 42-86 of SEQ ID NO: 4)),
Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg-Val-Gly-Pro—Thr-Gly-Glu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 11 (corresponding to the amino acid position 44-67 and 92-112 of SEQ ID NO: 2 or the amino acid position 18-41 and 66-86 of SEQ ID NO: 4)), Gly-Leu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 43 (corresponding to the amino acid position 41-43 and 92-112 of SEQ ID NO: 2)),
Gly-Leu-Lys-Gly-Asp-Met-Gly-Asp-Lys-Gly-Gln-Lys-Gly-Ser-Val-Gly-Arg-His-Gly-Lys—Ile-Gly-Pro-Ile-Gly-Ser-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 42 (corresponding to the amino acid position 41-43 and 68-112 of SEQ ID NO: 2)), or
Gly-Leu-Lys-Gly-Asp-Ala-Gly-Glu-Lys-Gly-Asp-Lys-Gly-Ala-Pro-Gly-Arg-Pro-Gly-Arg—Val-Gly-Pro-Thr-Gly-Glu-Lys-Gly-Glu-Lys-Gly-Asp-Ser-Gly-Asp-Ile-Gly-Pro-Pro-Gly-Pro-Asn-Gly-Glu-Pro-Gly-Leu-Pro (SEQ ID NO: 44 (corresponding to the amino acid position 41-67 and 92-112 of SEQ ID NO: 2)).
9. A base sequence encoding the protein according to claim 7 or 8.
10. A protein comprising an amino acid sequence of:
Met-Arg-Gly-Asn-Leu-Ala-Leu-Val-Gly-Val-Leu-Ile-Ser-Leu-Ala-Phe-Leu-Ser-Leu—Leu-Pro-Ser-Gly-His-Pro-Gln-Pro-Ala-Gly-Asp-Asp-Ala-Cys-Ser-Val-Gln-Ile-Leu-Val-Pro (SEQ ID NO: 53 (corresponding to the amino acid position 1-40 of SEQ ID NO: 2)) at an N-terminal of the sequence (Gly-Xaa-Yaa)n of the protein according to claim 7 or 8 in addition thereto.
11. A protein comprising a sequence of: Met-Trp-Trp-Val-Pro-Pro-Ser-Pro-Tyr-Gly-Cys-Leu-Pro-Cys-Ala-Leu-Pro (SEQ ID NO: 54 (corresponding to the amino acid position 1-17 of SEQ ID NO: 4)) at an N-terminal of the sequence (Gly-Xaa-Yaa)n of the protein according to claim 7 or 8 in addition thereto.
12. A base sequence encoding the protein according to claim 10 or 11.
13. A protein comprising an amino acid sequence consisting of 197 amino acids set out in the amino acid position 1-197 of SEQ ID NO: 6, or a protein consisting of an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6, or a derivative or a fragment thereof.
14. A base sequence set out in SEQ ID NO: 55 (corresponding to a base sequence set out in the base position 141-731 of SEQ ID NO: 5), a base sequence encoding an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-197 of SEQ ID NO: 6.
15. A protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 8, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8, or a derivative or a fragment thereof.
16. A base sequence set out in SEQ ID NO: 56 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 7), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 8.
17. A protein comprising an amino acid sequence consisting of 221 amino acids set out in the amino acid position 1-221 of SEQ ID NO: 10, or a protein consisting of an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10, or a derivative or a fragment thereof.
18. A base sequence set out in SEQ ID NO: 57 (corresponding to a base sequence set out in the base position 141-803 of SEQ ID NO: 9), a base sequence encoding an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-221 of SEQ ID NO: 10.
19. A protein comprising an amino acid sequence consisting of 271 amino acids set out in the amino acid position 1-271 of SEQ ID NO: 13, or a protein consisting of an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13, or a derivative or a fragment thereof.
20. A base sequence set out in SEQ ID NO: 58 (corresponding to a base sequence set out in the base position 157-969 of SEQ ID NO: 12), a base sequence encoding an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-271 of SEQ ID NO: 13.
21. A protein comprising an amino acid sequence consisting of 223 amino acids set out in the amino acid position 1-223 of SEQ ID NO: 37, or a protein consisting of an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37, or a derivative or a fragment thereof.
22. A base sequence set out in SEQ ID NO: 59 (corresponding to a base sequence set out in the base position 265-933 of SEQ ID NO: 36), a base sequence encoding an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-223 of SEQ ID NO: 37.
23. A protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 39, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39, or a derivative or a fragment thereof.
24. A base sequence set out in SEQ ID NO: 60 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 38), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 39.
25. A protein comprising an amino acid sequence consisting of 247 amino acids set out in the amino acid position 1-247 of SEQ ID NO: 41, or a protein consisting of an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 having deletion, substitution or addition of one or several amino acids therein and having an equal property to that of the protein having an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41, or a derivative or a fragment thereof.
26. A base sequence set out in SEQ ID NO: 61 (corresponding to a base sequence set out in the base position 265-1005 of SEQ ID NO: 40), a base sequence encoding an amino acid sequence set out in the amino acid position 1-247 of SEQ ID NO: 41 or a fragment thereof, or a base sequence that hybridizes to any one of said base sequences or a base sequence complementary thereto under a stringent condition and encodes a protein having an equal property to that of the protein having an amino acid sequence set out in amino acid position 1-247 of SEQ ID NO: 41.
27. A vector comprising a base sequence according to claim 2, 4, 6, 9, 12, 14, 16, 18, 20, 22, 24 or 26.
28. A transformed cell carrying a base sequence according to claim 2, 4, 6, 9, 12, 14, 16, 18, 20, 22, 24 or 26 in a manner to allow the expression.
29. A process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to claim 2, 4, 6, 9, 12, 14, 16, 18, 22, 24 or 26; and harvesting thus produced hCL-L2s protein.
30. A process for the production of a protein which comprises the steps of: culturing a cell transformed with the base sequence according to claim 20; and harvesting thus produced mCL-L2s protein.
31. The process according to claim 29 or 30 wherein said cell is Escherichia coli, an animal cell or an insect cell.
32. A transgenic non-human animal having an altered expression level of CL-L2s gene.
33. A knockout mouse wherein a function of mCL-L2s gene is deficient.
34. An antibody to the protein according to claim 1, 3, 5, 7, 8, 10, 11, 13, 15, 17, 19, 21, 23 or 25, or a fragment thereof.
35. The antibody according to claim 34, which is a polyclonal antibody, a monoclonal antibody or a peptide antibody.
36. A process for measuring a CL-L2s protein or a fragment thereof on the basis of an immunological binding between the antibody according to claim 34 or 35 and the CL-L2s protein or a fragment thereof.
37. An agonist that stimulates a function of the protein according to claim 1, 3, 5, 7, 8, 10, 11, 13, 15, 17, 19, 21, 23 or 25.
38. An antagonist that inhibits a function of the protein according to claim 1, 3, 5, 7, 8, 10, 11, 13, 15, 17, 19, 21, 23 or 25.
39. A process for screening a drug wherein the protein according to claim 1, 3, 5, 7, 8, 10, 11, 13, 15, 17, 19, 21, 23 or 25 is used.
40. A drug which is obtained by the process for the screening according to claim 39.
US10/258,105 2000-04-21 2001-04-23 Novel collectins Abandoned US20030158382A1 (en)

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US12/334,052 US7985833B2 (en) 2000-04-21 2008-12-12 Collectin
US13/087,989 US20120015400A1 (en) 2000-04-21 2011-04-15 HCL-K1 Polypeptide Which Offers Collectin Activity
US13/160,091 US8604162B2 (en) 2000-04-21 2011-06-14 Collectin

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PCT/JP2007/057632 Continuation-In-Part WO2007116925A1 (en) 2000-04-21 2007-04-05 hCL-K1 POLYPEPTIDE HAVING COLLECTIN ACTIVITY
US12/334,052 Continuation US7985833B2 (en) 2000-04-21 2008-12-12 Collectin
US29608509A Continuation-In-Part 2000-04-21 2009-07-22

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US10/258,105 Abandoned US20030158382A1 (en) 2000-04-21 2001-04-23 Novel collectins
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090305341A1 (en) * 2006-04-05 2009-12-10 Fuso Pharmaceutical Industries, Ltd HCL-K1 Polypeptide Which Offers Collectin Activity
US20100203129A1 (en) * 2009-01-26 2010-08-12 Egalet A/S Controlled release formulations with continuous efficacy
US11466060B2 (en) 2017-03-31 2022-10-11 Green Rice Sweden Ab Carbohydrate producing plant material

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3059401A (en) 2000-02-14 2001-08-20 Fuso Pharmaceutical Ind Novel scavenger receptors
WO2001081401A1 (en) * 2000-04-21 2001-11-01 Fuso Pharmaceutical Industries, Ltd. Novel collectins
WO2007010623A1 (en) * 2005-07-22 2007-01-25 Phg Corporation Novel polypeptide and method for producing the same
CA2850450A1 (en) 2011-12-23 2013-06-27 Basf Plant Science Company Gmbh Genes to enhance disease resistance in crops
BR112015000272A2 (en) * 2012-07-13 2017-06-27 Unilever Nv packaged food concentrate, process for preparing a packaged food concentrate and use of concentrated food composition
CN120660000A (en) * 2023-03-23 2025-09-16 积水医疗株式会社 Method for determining lung surfactant protein D, determination kit, monoclonal antibody and cell

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6787639B1 (en) * 1998-01-23 2004-09-07 Fuso Pharmaceutical Industries, Ltd. Collectin

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4873191A (en) 1981-06-12 1989-10-10 Ohio University Genetic transformation of zygotes
JPS6336786A (en) 1986-07-31 1988-02-17 Teijin Ltd Nucleic acid base sequence of antibody light chain manifestation type, plasmid, cell and chimera antibody light chain
JP2868777B2 (en) 1987-08-20 1999-03-10 チルドレンズ・ホスピタル・コーポレイション Human mannose binding protein
US5270199A (en) 1987-08-20 1993-12-14 The Children's Medical Center Corporation Human mannose-binding protein
GB8720833D0 (en) 1987-09-04 1987-10-14 Celltech Ltd Recombinant dna product
JP3739009B2 (en) 1989-11-07 2006-01-25 俊雄 高橋 Mouse antibody V region gene fragment and method for preparing chimeric antibody using the gene fragment
GB8928874D0 (en) 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
GB9009549D0 (en) 1990-04-27 1990-06-20 Celltech Ltd Recombinant antibody and method
GB9009548D0 (en) 1990-04-27 1990-06-20 Celltech Ltd Chimeric antibody and method
GB9014932D0 (en) 1990-07-05 1990-08-22 Celltech Ltd Recombinant dna product and method
JPH04330295A (en) 1990-12-28 1992-11-18 Mitsubishi Kasei Corp Mouse-human chimeric antibody
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
JP4124480B2 (en) 1991-06-14 2008-07-23 ジェネンテック・インコーポレーテッド Immunoglobulin variants
CA2078539C (en) 1991-09-18 2005-08-02 Kenya Shitara Process for producing humanized chimera antibody
GB9120467D0 (en) 1991-09-26 1991-11-06 Celltech Ltd Anti-hmfg antibodies and process for their production
JP2524939B2 (en) 1991-12-03 1996-08-14 株式会社ニチレイ Enhancer and its application
CA2128544A1 (en) 1992-12-10 1994-06-23 Celltech Therapeutics Limited Humanised antibodies directed against a33 antigen
JPH06336786A (en) 1993-05-26 1994-12-06 Matsushita Electric Works Ltd Device for attachment of ceiling panel for bathroom
CA2163345A1 (en) 1993-06-16 1994-12-22 Susan Adrienne Morgan Antibodies
WO2002008284A2 (en) * 2000-07-20 2002-01-31 Genentech, Inc. Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
JP4108760B2 (en) 1997-02-21 2008-06-25 ジェネンテック，インコーポレーテッド Antibody fragment-polymer conjugate and humanized anti-IL-8 monoclonal antibody
JP3233057B2 (en) 1997-02-27 2001-11-26 日本鋼管株式会社 Measuring method of spangle size of hot-dip coated steel sheet
ATE501170T1 (en) 1997-04-07 2011-03-15 Genentech Inc METHOD FOR PRODUCING HUMANIZED ANTIBODIES BY RANDOMIZED MUtagenesis
WO1998055614A2 (en) 1997-06-04 1998-12-10 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
IL139686A0 (en) * 1998-06-02 2002-02-10 Genentech Inc Secreted and transmembrane polypeptides and nucleic acids encoding the same
WO2000018922A2 (en) * 1998-10-01 2000-04-06 Incyte Genomics, Inc. Human carbohydrate-associated proteins
AU2495200A (en) * 1999-03-08 2000-09-28 Genentech Inc. Compositions and methods for the treatment of tumor
CA2383254A1 (en) * 1999-06-02 2000-12-07 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
WO2001081401A1 (en) * 2000-04-21 2001-11-01 Fuso Pharmaceutical Industries, Ltd. Novel collectins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6787639B1 (en) * 1998-01-23 2004-09-07 Fuso Pharmaceutical Industries, Ltd. Collectin

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090305341A1 (en) * 2006-04-05 2009-12-10 Fuso Pharmaceutical Industries, Ltd HCL-K1 Polypeptide Which Offers Collectin Activity
US20100203129A1 (en) * 2009-01-26 2010-08-12 Egalet A/S Controlled release formulations with continuous efficacy
US11466060B2 (en) 2017-03-31 2022-10-11 Green Rice Sweden Ab Carbohydrate producing plant material

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PT1283214E (en) 2007-03-30
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US20090275740A1 (en) 2009-11-05
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ATE353913T1 (en) 2007-03-15
AU4884001A (en) 2001-11-07
JPWO2001081401A1 (en) 2004-04-15
DE60126602D1 (en) 2007-03-29
US7985833B2 (en) 2011-07-26
CA2406884A1 (en) 2002-10-18
AU2001248840B2 (en) 2005-06-09
EP1283214A1 (en) 2003-02-12
US8604162B2 (en) 2013-12-10
EP1881004B1 (en) 2012-07-25
WO2001081401A1 (en) 2001-11-01
EP1881004A1 (en) 2008-01-23

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