WO1996001277A1 - METHOD FOR IMMUNOASSAYING SOLUBLE Fas ANTIGEN AND KIT THEREFOR - Google Patents

Abstract

A kit and a method for immunoassaying soluble Fas antigens present in a body fluid such as serum, whereby the quantity of soluble Fas antigens present in the body fluid of a patient with autoimmune diseases such as rheumatism or SLE, which has been difficult to determine heretofore, can be determined readily with high accuracy and sensitivity, and a large quantity of specimens can be assayed at once. The invention also provides a soluble Fas antigen as a soluble active substance which is very useful as a pure standard to be used in the above method and kit and as an immunizing antigen to be used for producing monoclonal antibodies against the soluble region of a Fas antigen.

Description

 . Specification

 Immunoassay method for soluble Fas antigen and kit for its measurement

 〔Technical field〕

 The present invention relates to a kit for immunological measurement of a soluble Fas antigen, a method for immunological measurement of a soluble Fas antigen, the kit for the measurement, a chimeric soluble Fas antigen that can be used in the measurement method, and a DNA encoding the same. The present invention relates to an expression vector containing the DNA, a transformed cell transformed with the expression vector, and a method for producing a chimeric soluble Fas antigen by culturing the cell. Furthermore, the present invention relates to a monoclonal antibody reactive with the chimeric soluble Fs antigen, and a fused cell producing the monoclonal antibody.

 (Background technology)

 In multicellular organisms, many cells are eliminated by cell death during ontogenesis. It is speculated that this cell death is caused by a predetermined program. In addition, in the adult, on the one hand, if the cell number increases due to cell division, on the other hand, cell death occurs, and it is thought that the total cell number is maintained in balance. Wyllie et al. Observed the morphology of dying cells by electron microscopy and categorized cell death into two morphologies: necrosis and apoptosis (Wyllie AH et al, Int. Cytol. 68, 251-306, 1980).

In the case of necrosis, the permeability of cell membranes increases at an early stage, organelles such as nuclear mitochondria swell, and eventually lysosomes are destroyed, and cells are destroyed by released proteases etc. I do. On the other hand, in the case of apoptosis, no major change in the structure of mitochondrial perisome is observed, and chromosomes condense in the nucleus and cytoplasm shrink in the early stages. At the same time, the nucleus may be fragmented into several parts, or a bubble-like structure may form on the cell surface, after which the cell may be divided into minicells called apoptotic bodies. Passive death, which is caused by external causes, so to speak, is mainly caused by necrosis, whereas preprogrammed cell death seen during development, differentiation or tissue turnover is thought to be caused by apoptosis. _N The protein present on the surface of living cells, which is thought to be closely related to the control of cell death called apoptosis or programmed cell death, is called the Fas antigen and penetrates the lipid bilayer of the cell membrane once or several times. It is a transmembrane protein composed of three main regions: an extracellular region (soluble region), a transmembrane region and a cytoplasmic region. The amino acid sequence of the mouse and human Fas antigens and the cDNA sequence encoding the amino acid sequence have already been elucidated (Cell. 66. 233-243, 1991, Itunaoto et al., J. Immunol., Vol. 148, No. 4, pp. 1274-1279, 1992, Watanabe (Fukunaga) Rie et al., J. BioL Chem., Vol. 267, No. 15, 10709-10715, 1992, Ahm (Alexander Oehm) et al., J. Exp. Med., Vol. 169. 1747-1756. 1989, Yoneharasin et al., Proc. Natl. Acad. Sci. USA. 87, 9620-9624. 1990, Kobayashi. Nobuyuki et al., Science, Vol. 245, p. 301-305. 1989. Traus (Bernhard C. Trauth) et al., J. Immunol.. Vol. 149. No. 10. 3166-3173, 1992, Dyne ( Jens Dhein) et al., The Lancet, Vol. 335. 497-500, 1990, Klaus-Michael Debatin et al., J. Immunol.. 149, 11, 3753-3758. 1992. Miyazaki Toshio et al., Genomics. Vol.14. 179-1 P. 80. See 1992, Peter Lichter et al.).

 Yonehara et al. Produced a monoclonal antibody against the cell surface antigen of human FS-7 cells and obtained an anti-Fas antibody, which showed activity to kill cells expressing the Fs antigen ( J. Exp. Ed., 169: 1747-1756, 1989). In addition, since the cell death showed characteristics of apoptosis by observation with an electron microscope, it was concluded that the F ss antigen mediates cell death by apoptosis. Separately, Traauth et al. Have obtained an APO-1 antibody, a monoclonal antibody that has properties resembling anti-Fas antibodies and has an activity to kill cells (Science, Vol. 245. 301). -P.305, 1989). These two antibodies are not only common in inducing cell death, but also have very similar antigen distribution and molecular weight, and have been shown to be the same.

As a result of structural analysis, the Fas antigen has a structure very similar to tumor necrosis factor (TNF) receptor and nerve growth factor (NGF), and a new type that causes cell death by receiving an external signal. TN F / It is one of the receptors that form the NGF receptor family.

 Recently, abnormalities in the Fas gene have been observed in mice with autoimmune symptoms (MRL I prZI pr mice), and high levels of apoptosis in lymphocytes have been observed in HIV-infected individuals. It has been suggested that the Fas antigen is related, and attention has been paid to the relationship between the expression of the Fas antigen and diseases such as autoimmune diseases.

 (Chengt. J. et al, Science, 263, 1759-1762, 1994) reported that an autoimmune disease [rheumatic disease] was used to examine the association between abnormal transcription of these Fas antigens and autoimmune diseases. (Rheumatoid Arthritis; RA). SL ECSystemic Lupus erythematosus)] Intracellular cDNAs were extracted from patients and healthy subjects and examined. In both cases, a normal 1167 bp cDNA encoding the full-length Fas antigen was obtained. In addition, they found that a small cDNA of 1104 bp was found. Furthermore, when the small cDNA of 114 bp was compared with that of cDNA of 1167 bp, the small cDNA (FasATM) was found to be lacking the hydrophobic transmembrane domain (TM, transmembrane domain). ) Encodes a Fa s antigen (hereinafter referred to as "soluble Fa s antigen j") that is soluble in blood, serum, plasma, and body fluids such as urine, saliva, and bone marrow.

Chengt et al. Showed that treating mice with soluble Fas antigen increased the number of CD4 and CD8-only positive and double-negative cells and reduced the number of double-positive cells in thymocytes. Because the total number of cells in the spleen cysts increases, and the amount of soluble Fas antigen in serum in SLE patients is about twice as large as that in healthy individuals, He developed the etiology that overproduction of antigens inhibited apoptosis in the thymus and peripheral tissues and did not properly eliminate autoreactive lymphocyte clones, leading to the development of autoimmune diseases. . Therefore, measuring the amount of soluble Fa s antigen in body fluids such as serum is important for predicting, diagnosing and treating various diseases that may be related to the dynamics of Fa s antigen such as autoimmune diseases. It is useful for establishing and determining the effects of treatment, and there is a keen need to establish the measurement method and measurement reagents (kits). Methods for detecting and measuring soluble Fas antigens in body fluids such as serum include the Western blot method (WB method), radioimmunoassay (Radioimmunoassay ^ RIA), and enzyme immunoassay. The method (Enzyme Immunoassay: EIA method or Enzyme-linked Immunosolbent Assay: ELISA method) can be considered. However, the W.B. method has high sensitivity and high specificity, but lacks quantitativeness, and is not suitable for processing a large number of samples at once. In addition, since the RIA method uses radioactive substances as labeling substances, it has the disadvantage that it can only be used by qualified persons in controlled facilities. In general, the EIA method or the ELISA method does not have the above-mentioned problems and can be measured easily and with high sensitivity, but until now, soluble Fas antigens in body fluids such as serum using such a method have been used. A simple measurement method with high accuracy and sufficient sensitivity is not known.

 In order to measure (quantify) soluble Fas antigens contained in body fluids such as serum, a calibration method is needed to confirm the sensitivity or accuracy of the measurement method and to indirectly determine the soluble Fas antigen content from the measured values. In order to create a line, it is necessary to obtain a purified soluble Fas antigen as a standard. As a standard substance for measuring the amount of soluble Fs antigen, it is desirable to use a purified protein product containing at least the Fs antigen soluble region (extracellular region) soluble in serum or the like.

 However, even with the use of genetic technology that has been widely used in mass production of proteins, the soluble regions of many transmembrane proteins such as the Fas antigen can be easily converted into soluble activators at the in-vitro mouth. In addition, it is very difficult to obtain them in large quantities, and it has hardly been achieved.

As a method for producing a soluble region (extracellular region) of a membrane-permeable protein, for example, a DNA sequence encoding a soluble region (extracellular region) of a desired transmembrane protein is converted to an immunoglobulin heavy chain ( A method of expressing a recombinant chimeric soluble protein using a DNA sequence linked to a DNA sequence encoding a constant region (Hc chain) of the H chain is known (Nature. Vol. 337, 525). -531. 1989. Capon et al., J. Exp. Med., Linsley et al., International Patent Application Publication. W093 / 00431, International Patent Application Publication W091 / 10438, etc.). However, in this method, the expression efficiency of the target protein is extremely low, and the Fc region of the expressed recombinant quinula-soluble protein has an ability to bind to immunoglobulin Fc receptor pig. However, there were major drawbacks such as the inability to accurately test and measure the function and biological activity of the target protein region.

 Therefore, the development of a method capable of easily and efficiently producing the extracellular region of a transmembrane protein such as the Fas antigen as a soluble active substance in vitro while maintaining an intact three-dimensional structure has been proposed. Immunization for producing a monoclonal antibody against the soluble region, which is important as a ligand recognition site of the transmembrane protein, as well as mass-producing the standard substance used in the measurement method and the measurement kit of the present invention. It enables mass production of antigens, and development of such a method is eagerly awaited.

 On the other hand, mouse hematopoietic cells express a transmembrane protein called AIC2A in their cell membrane. AIC2A is an iS subunit that constitutes a receptor for mouse IL-13 (hereinafter, also referred to as "mouse IL-13 receptor yS subunit AIC2AJ"), and forms a heterodimer with various subunits. As a high-affinity receptor for mouse IL-13 [The EMBO Journal. Vol. 5, No. 5, pp. 1875-1884. 1992, Haruya Kahiko et al., Blood. No. 79. No. 4 Vol. 80, No. 1, pp. 84-90, 1992, Kirato Muratoshio et al., The EMBO Journal. Vol. 10, No. 10, 2833-2838. 1991. Taka Kisatoshi et al., International Immunology. Vol. 3, No. 12, 1231-1242. See J. Schreurs et al.].

AIC 2 A, especially the amino acid sequence constituting the extracellular region thereof and the cDNA sequence encoding the amino acid sequence have already been elucidated [Science, Vol. 247. 324-327. 1990, Natl. Acad. Sci. USA, Vol. 87, pp. 5459-5463, 1990, Daniel (Daniel M. Gorman) et al., J. Bio. Chem., Vol. 267, No. 2. 979- 983, 1992. See Huey-Mei Wang et al.]. According to these reports, mouse IL-13 receptor / 3 subunit AIC2A is a transmembrane protein. It is noteworthy that, despite being a protein, its extracellular domain protein is efficiently expressed in cells of higher animals using the cDNA sequence encoding the region.

 [Disclosure of the Invention]

 An object of the present invention is to provide an immunoassay kit for measuring a soluble Fas antigen in a body fluid such as serum which could not be measured conventionally, and an immunoassay method for a soluble Fas antigen. That is. Another object of the present invention is to provide a chimeric soluble Fas antigen that can be used in the kit and method for immunoassay of the soluble Fas antigen, a DNA encoding the same, and an expression containing the DNA. An object of the present invention is to provide a vector, a transformed cell transformed with the vector, and a method for producing a chimeric soluble Fas antigen by culturing the cell. Furthermore, an object of the present invention is to provide a monoclonal antibody having reactivity with the chimeric soluble Fs antigen, and a fused cell producing the monoclonal antibody.

 Under the above-mentioned technical background, the present inventors have proposed a highly accurate, highly sensitive and simple immunoassay method for a soluble Fas antigen in a body fluid such as serum using an ELISA method (or an EIA method). After extensive research on immunoassay kits, it was found that a plurality of anti-Fas monoclonal antibodies produced by the present inventors differed from soluble Fas antigen and intact Fas antigen in body fluids such as serum, respectively. It was found that it has reactivity.

Further, it is useful as a standard substance used in the immunoassay method and the immunoassay kit for the soluble Fas antigen, or as an immunizing antigen used for producing a monoclonal antibody against the Fas antigen. It is possible to easily and efficiently express the extracellular region of the Fas antigen, a transmembrane protein, at the mouth of invit using a gene recombination technique as a soluble activator while maintaining its intact three-dimensional structure. As a result of diligent research on possible methods, DNA encoding the amino acid sequence constituting the extracellular domain of the Fas antigen and mouse IL-13 receptor 13 subunit AIC 2 DN encoding the amino acid sequence constituting the extracellular domain of AIC 2A By using the chimeric DNA consisting of A, the soluble region of the Fas antigen It has been found that it is possible to easily and mass-produce a soluble active substance in the form of a protein (hereinafter referred to as “chimeric soluble Fas antigen”).

 As a result of further studies based on these findings, the chimera as a chimeric protein between the soluble region of the Fas antigen and the extracellular region of mouse IL-13 receptor / 5 subunit AIC2A prepared by the present inventors. Soluble in body fluids such as serum can be obtained by using a sandwich ELISA using a soluble Fas antigen as a standard substance and one or more monoclonal antibodies arbitrarily selected from the plurality of anti-Fas monoclonal antibodies. The present inventors have found that the Fas antigen can be easily measured with high accuracy and high sensitivity, and have completed the present invention.

That is, a first aspect of the present invention relates to a solid-phased anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fas antigen to an insoluble support, and a standard substance. This is a measurement kit for immunologically measuring a soluble Fas antigen, which comprises a soluble Fas antigen. Specifically, a measurement for immunologically measuring a soluble Fas antigen, which further comprises a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen. Kit. More specifically, 1) the soluble Fas antigen as a standard substance is composed of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-13 receptor S subunit AIC2A or immunoglobulin weight. A kit for immunologically measuring a soluble Fas antigen, which is characterized as being a chimeric soluble Fas antigen comprising a constant region of the chain, 2) an anti-Fas monoclonal antibody bound to an insoluble support Is a monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CHI1. 3) Second anti-Fas monoclonal antibody consists of hybridoma clones VB3, CBE, WB 3. ZB4, UB2, AX6, JAE and CHI1 From the group Soluble F the as antigen immunologically measuring kit for measuring to Toku徵 that from the barrel High Priestess dormer is a monoclonal antibody which is produced or, 4) labeled second anti F the as monochromator Immunizing a soluble Fas antigen characterized in that the ronal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, 8-D-galactosidase, microperoxidase, alkaline phosphatase, and biotin. This is a measurement kit for performing a biological measurement.

 A second aspect of the present invention is a method for immunologically measuring a soluble Fs antigen, which comprises the following steps a) to c) and d) to f).

a) A soluble Fas antigen as a standard was reacted with an immobilized anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fas antigen to an insoluble support. Later

b) reacting a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen;

c) Measure the amount of labeling of the reaction product

Creating a calibration curve by

d) After reacting the sample with an immobilized anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fs antigen to an insoluble support,

e) reacting a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen,

 f) Measure the amount of labeling of the reaction product,

A step of quantifying a soluble F ss antigen contained in the sample from the calibration curve.

Specifically, 1) the soluble Fas antigen as the standard substance is composed of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-13 receptor 3 subunit AIC2A or the immunoglobulin heavy chain. A chimeric soluble Fas antigen comprising a constant region, and a method for immunologically measuring a soluble Fas antigen, 2) an anti-Fas monoclonal antibody bound to an insoluble support, comprising a hybridoma clone VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CH11 Immunoassay method for soluble Fas antigen characterized by being a monoclonal antibody produced from a hybridoma selected from the group consisting of CH11, 3) Second Anti-F As a monoclonal antibody, wherein the monoclonal antibody is a monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CHI1. A method for immunoassay of Fas antigen, or 4) the second labeled anti-Fas monoclonal antibody is selected from the group consisting of peroxidase, yS-D-galactosidase, microperoxysidase, lipophosphatase, and biotin. This is an immunoassay method for a soluble Fas antigen, which is a monoclonal antibody labeled with a selected labeling substance.

 The third aspect of the present invention is a chimeric soluble Fas antigen comprising the extracellular region of human or mouse Fas antigen and the extracellular region of mouse IL13 receptor A-subunit AIC2A. Specific examples include a chimeric soluble Fas antigen having the amino acid sequence shown in SEQ ID NO: 11 or 19 in the sequence listing.

 A fourth aspect of the present invention is a DNA encoding the chimeric soluble Fas antigen. Specific examples include a DNA having a DNA sequence shown in SEQ ID NO: 10 or 18 in the sequence listing.

 A fifth aspect of the present invention is an expression vector containing a DNA encoding the chimeric soluble Fas antigen. Specific examples include an expression vector containing the DNA sequence shown in SEQ ID NO: 10 or 18 in the sequence listing.

 A sixth aspect of the present invention is a transformed cell transformed with the expression vector. Specifically, a cell line identified by International Accession No. FERM BP-4436 or 4437 can be mentioned.

 A seventh aspect of the present invention is a method for producing a chimeric soluble Fas antigen by culturing the transformed cell. More specifically, there is provided a method for producing a chimeric soluble Fs antigen, which comprises culturing the transformed cells in a medium and obtaining proteins produced in the culture supernatant.

An eighth aspect of the present invention is a fused cell obtained by cell fusion between an antibody-producing cell obtained from a mammal immunized with the chimeric soluble Fas antigen and a mammalian myeloma cell line. Specifically, International Deposit No. FERM BP—4394, 4667 Or the cell line identified in 4756.

 A ninth aspect of the present invention is a monoclonal antibody reactive with the chimeric soluble Fs antigen, specifically, a monoclonal antibody produced by the fused cells.

 [Brief description of drawings]

 FIG. 1 is a diagram showing a calibration curve in a Sandwich ELISA method using a chimeric soluble human Fas antigen as a standard substance (standard solution). The vertical axis represents the absorbance, and the horizontal axis represents the concentration (ng / nil) of the standard chimeric soluble human Fas antigen.

 FIG. 2 is a diagram showing a calibration curve in the Sandwich ELISA method using a chimeric soluble human Fas antigen as a standard substance (standard solution). The vertical axis represents the absorbance, and the horizontal axis represents the concentration (ngZml) of the standard chimeric soluble human Fas antigen.

 FIG. 3 is a restriction map of plasmid pCEV4.

 FIG. 4 is a restriction map of plasmid pME18S.

 FIG. 5 is a construction process diagram of plasmid pME18S / hFas · EXT. Figure 6 is a construction process diagram of the plasmid pMEl8ShFas · EXT—AIC2A · EXT.

 Figure 7 shows SDS-polyacrylamide gel electrophoresis for confirming the expression of hFas · EXT-AlC2A · EXT (chimeric soluble human Fas antigen) in the culture supernatant. FIG.

 Figure 8 shows cell death of Fa s antigen-expressing cells induced by anti-human Fa s antibody CHI 1 by apoptosis by soluble h Fa s -AIC 2A protein (Qin) and soluble AI C2A protein (□). It is a figure which shows the suppression effect. The vertical axis represents the viable cell rate (%), and the horizontal axis represents the dilution rate.

 Figure 9 is a construction process diagram of plasmid pME18S / mFas · EXT. FIG. 10 is a process diagram of the construction of plasmid pME18SmFas · EXT—AIC2A · EXT.

Figure 11 shows SDS-polyacrylamide gel electrophoresis analysis to confirm the expression of mFas'EXT-AIC2A · EXT (chimeric soluble mouse Fas antigen) in the culture supernatant. FIG. FIG. 12 shows rat anti-mouse Fas monoclonal antibodies RMF2, RF6, RMF9 and RMF13 (each indicated by a dotted line) and control monoclonal antibodies, anti-rat IgG or anti-rat IgM ( Each is shown by a solid line.) Is a diagram showing the reactivity to L5178Y that does not express Fas antigen and L5178Y that overexpresses mouse Fas antigen. The vertical axis represents the number of cells, and the horizontal axis represents the amount of the monoclonal antibody bound to the cell surface expressed by the fluorescence intensity.

 FIG. 13 shows hamster anti-mouse Fas monoclonal antibodies RK-8, SK-8, C6-1 and P4-4 (each indicated by a dotted line) and a control monoclonal antibody, anti-hamster IgG (each a solid line). Fig. 4 shows the reactivity of L5178Y not expressing Fas antigen and L5178Y overexpressing mouse Fas antigen. The vertical axis represents the number of cells, and the horizontal axis represents the amount of the monoclonal antibody bound to the cell surface represented by the fluorescence intensity.

 [Detailed description of the invention]

 Hereinafter, the present invention will be described in more detail by clarifying the meanings of phrases used in the present invention.

 The term "I" Fas antigen J in the present invention means, unless otherwise specified, a mammalian Fas antigen such as human, mouse, rat, guinea pig, hamster, rabbit, dog or monkey, and preferably human Mouse or rat Fas antigen, particularly preferably human Fas antigen.

In the measurement kit and the measurement method of the present invention, "in the case of the immobilized anti-Fas monoclonal antibody J bound to the insoluble support J, It is used to be supported on the support by adsorption or chemical bonding. For example, water such as plastic represented by polystyrene resin, polycarbonate resin, silicone resin or nylon resin, glass, etc. It is made of an insoluble substance and has an internal volume for adding a sample such as serum, etc. Any known material can be used as long as such a material is used. Considering the point of simultaneous processing of plastics, for example, a plastic plate having a large number of wells such as a 96-well microplate Preferably, a rate is used. The term "immobilized anti-Fas monoclonal antibody j" means an anti-Fas monoclonal antibody that is supported on the "insoluble support" by physical adsorption or chemical bonding. By reacting a sample such as serum with the “immobilized anti-Fas monoclonal antibody”, the soluble Fas antigen contained in the sample is trapped by the present antibody.

Regarding the “labeled second anti-Fas monoclonal antibody” in the measurement kit and the measurement method of the present invention, the “labeling substance” used for labeling j is peroxidase, 1D-galactosidase, micropel Enzymes such as oxidase or alkaline phosphatase;] I, ³ I, radioisotopes such as C or tritium, and antibodies or antigens such as biotin. Examples of commonly used labeling substances are exemplified. “When using piotin as the labeling substance j, a method of labeling with piotin and reacting with avidin peroxidase is used. According to this method, measurement can be performed with higher sensitivity and sensitivity. In view of the convenience of the kit, it is preferable to use an enzyme such as peroxidase, β-D-galactosidase, microperoxidase, or calcium phosphatase, or biotin. The “monoclonal antibody” means an anti-Fas monoclonal antibody labeled with a labeling substance as described above. The soluble anti-Fas antigen trapped by the immobilized anti-Fas monoclonal antibody described above is allowed to react with the second anti-Fas monoclonal antibody J, which has been labeled, and specifically bound to the solid anti-Fas monoclonal antibody. It is possible to detect an antigen-antibody complex of a simulated anti-Fas monoclonal antibody and a soluble Fs antigen. The second anti-Fas monoclonal antibody J may be the same monoclonal antibody as the above-mentioned Γthe immobilized anti-Fas monoclonal antibody or may be a different monoclonal antibody, but is preferably used. Is a monoclonal that can specifically bind to an antigenic determinant at a position different from the antigenic determinant of the soluble Fas antigen to which the immobilized anti-Fas monoclonal antibody binds.

The second anti-Fas monoclonal antibody labeled with the immobilized anti-Fas monoclonal antibody usually recognizes different antigenic determinants of the soluble Fas antigen. If two identical antigenic determinants are present, they may recognize the same antigenic determinant. In this case, one of the determinants binds to the immobilized anti-Fas monoclonal antibody and the other one binds to the labeled second anti-Fas monoclonal antibody.

 The anti-Fas monoclonal antibody used as the “immobilized anti-Fas monoclonal antibody” or the “second anti-Fas monoclonal antibody” in the measurement kit and the measurement method of the present invention includes at least a bodily fluid such as serum. Any monoclonal antibody that can specifically bind to the soluble Fas antigen can be used. Specifically, for example, International Immunology. Vol. 6, No. 12, pp. 1849-1856, 1994, Journal of Experimental Medicine, Vol. 169, 1747-1756, 1989 and Cell, Vol. 66, 233. -243, report of Yonehara et al., 1991, and clones ZB4 (IgG), UB2 (IgG), which are anti-human Fas monoclonal antibody-producing hybrid clones described in Examples below. WB3

(I gG), VB3 (I gG), CBE (I gG), JAE (I gM), AX 6

(IgM) or anti-Fas monoclonal antibody produced from CHI1 (IgM) and the like.

 The measurement kit of the present invention contains at least a solid-phased anti-Fas monoclonal antibody bound to an insoluble support and a soluble Fas antigen as a standard substance, and preferably a labeled second anti-Fs It is characterized in that it contains a monoclonal antibody. According to the measurement kit, the ELISA method (E

The ISA method) or the ELISA method using the sandwich method (E1A method) (hereinafter, the Sandwich ELISA method) can be used to measure soluble Fas antigen in body fluids such as serum. It is possible. However, when measurement is performed by the ELISA method using a competitive method, it is necessary to obtain a large amount of purified Fas antigen that retains the intact structure of soluble Fas antigen present in blood. Obtaining large quantities of antigen is laborious. Therefore, it is preferable to use the sandwich ELISA method in the measurement of the soluble Fs antigen by the ELISA method.

The sandwich ELISA method of the present invention can be performed according to the purpose and conditions of measurement. The soluble Fas antigen is measured by combining at least two appropriately selected anti-Fas monoclonal antibodies and setting up a sandwich ELISA method. Specifically, it is characterized by using a solid-phased anti-Fas monoclonal antibody and a labeled second anti-Fas monoclonal antibody. The immobilized anti-Fas monoclonal antibody and the labeled second anti-Fas monoclonal antibody may be the same anti-Fas monoclonal antibody or different anti-Fas monoclonal antibodies. Alternatively, it is preferable to use a combination of two anti-Fas monoclonal antibodies that recognize and bind to different antigenic determinants of the soluble Fas antigen. If the same anti-Fas monoclonal antibody is used, two identical antigenic determinants are present at different positions on the soluble Fas antigen, and each monoclonal antibody binds to each of the two antigenic determinants. It means to do.

 An example of a specific combination of the “immobilized anti-Fas monoclonal antibody J” and the “labeled second anti-Fas monoclonal antibody” is as described above for the antibody-native hybrid monoclonal ZB4 (IgG). , UB2 (IgG), WB3 (IgG), VB3

(IgG) One or two hybridomas selected from CBE (IgG), JAE (IgM), AX6 (IgM), CHI1 (IgM), etc. are produced respectively.

One or two anti-Fas monoclonal antibodies can be immobilized or labeled before use. An example of a preferred combination includes, as a monoclonal antibody for preparing an immobilized anti-Fas monoclonal antibody, an anti-Fas monoclonal antibody obtained from clone CBE, and a labeled second anti-Fas monoclonal antibody. Anti-Fas monoclonal antibodies obtained from clone VB3 can be used.

The “chimeric soluble Fas antigen” of the present invention can be used as a standard substance for immunoassays and kits for immunoassay for soluble Fas antigens, or as an immunoassay used for the production of monoclonal antibodies against Fa sitogen. is useful as a sensitizing antigen _c the Γ chimeric soluble F the as antigens J is Kimeratanpa the soluble region and murine IL- 3 Resebu evening one Sabuyunitto AIC 2 a of F the as antigen using Yadenko engineering technique Or the soluble region of the Fas antigen and the constantity of the immunoglobulin heavy chain (H chain) It can be produced and used as a chimeric protein with a region (constant region: Fc region). Preferably, it is a chimeric protein of the soluble region of the Fas antigen and mouse IL-13 receptor / 3-subunit AIC2A, which is another subject of the present invention.

 This chimeric protein of the soluble region of the Fas antigen and the mouse IL-13 receptor 3 subunit A1C2A contains an amino acid sequence substantially constituting the extracellular region of the Fas antigen downstream of the expression promoter. An amino acid sequence that substantially constitutes the extracellular region of the encoding DNA and mouse 1L-1 / 3 receptor / 3 subunit AIC 2A An expression vector constructed by introducing a chimeric DNA consisting of the encoding DNA It can be obtained by transforming the host cell, culturing the resulting transformed cells in a medium, and producing the protein produced in the culture supernatant by simple hybridization. Specifically, it can be manufactured as follows.

 Total RNA is prepared from cells capable of producing a Fs antigen, and a cDNA library is prepared using these as a type II. The cells used here can be arbitrarily selected. If the cells produce human Fas antigen, for example, human T tumor cell line KT3, if mouse Fas antigen, mouse macrophage BAM 3 can be used.

 Next, a clone containing the cDNA encoding the Fs antigen is selected from the cDNA library, and a full-length cDNA encoding the Fs antigen is obtained from the clone. From the full-length cDNA, a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the Fs antigen is excised and introduced into an appropriate plasmid.

In the same manner as described above, a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the mouse interleukin-3 receptor subunit AIC2A or the constant region Fc of the immunoglobulin H chain is prepared in the same manner as described above. A cDNA fragment encoding a substantially constitutive amino acid sequence is obtained.

A mouse interleukin-13 receptor; 3 subunits AIC2A, with or without an inserted DNA sequence, downstream of a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the Fas antigen Substantially the extracellular area of A Fas antigen opened with a restriction enzyme to introduce a cDNA fragment encoding the constituent amino acid sequence or a cDNA fragment encoding the amino acid sequence substantially constituting the constant region Fc of the immunoglobulin H chain Said plasmid into which a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of said mouse has been introduced, and said extracellular region of said mouse interleukin-13 receptor 1; 3 subunit AIC2A A cDNA fragment encoding an amino acid sequence that constitutes an amino acid sequence or a cDNA fragment that encodes an amino acid sequence that substantially constitutes the constant region Fc of the immunoglobulin H chain. After modifying the DNA ends of the DNA sequence so that they can be linked to each other, each DNA fragment is subjected to DNA fragmentation using a DNA ligation kit in a conventional manner. The ligated to obtain Burasumi Dobe Kuta one chimeric protein with F the as antigens and AIC 2 A is unloaded capable braking produced.

 Next, a suitable host cell is transformed with the plasmid vector, the transformed cell is cultured in a medium, and a chimeric soluble Fs antigen as a chimeric protein is collected from the culture medium.

 The expression vector used in the production of the chimeric soluble Fas antigen may be selected from plasmids commonly used in test research or the industrial field, depending on the host cell to be cultivated, and according to the purpose. Can be selected and used. For example, pME18S, pCEV4, pEF-BOS, and PHSAPR-1 can be used.

These expression vectors are necessary for extracellular secretion of the chimeric soluble Fas antigen in addition to the DNA sequence encoding the amino acid sequence of the chimeric soluble Fas antigen in order to control the chimeric soluble Fas antigen so that it can be expressed. A DNA sequence encoding a novel signal peptide, and a promoter, which is a DNA sequence essential for the initiation of transcription of this DNA into messenger RNA (mRNA) by RNA polymerase, are co- DNA sequence encoding a terminator, which is a DNA sequence essential for the transcription termination control, and SD sequence (Shine-Dalgarno sequence) encoding a base sequence on mRNA that is a ribosome binding site. ; Shine 'Dalgarno sequence), translation initiation codon encoding the translation start point from mRNA to protein, translation termination codon encoding the translation end point from mRNA to protein, and necessary selection for selection of transformed cells Marker (selection marker) The DNA sequence has been introduced, and depending on the purpose, for example, a DNA sequence encoding a DNA replication origin essential for replication of this vector in a host cell, mRNA of DNA, etc. DNA sequence that encodes an enhancer, which is a DNA sequence for improving the transcription efficiency to DNA, and a polyadenylic acid signal sequence (poly A signal), which is a DNA sequence that improves the stability of mRNA. Such DNA sequences can be introduced.

 The expression promoter can be selected from promoters commonly used in test research or the industrial field, and can be used according to the host cell to be transformed and depending on the purpose. For example, SV40 promoter, LTR promoter, SRa promoter, EF-1α promoter, 3 actin promoter, imnoglobulin promoter and the like can be used.

 As the host cell, depending on the expression vector and the purpose, depending on the purpose, there are test studies, natural cells or artificially established recombinant cells, which are commonly used in the industrial field. It can be used by selecting from animal cells that are not limited to. For example, mouse-derived cells (COP, L, C127, Sp2 / 0, L5178Y and NS-1 etc.), rat-derived cells, hamster-derived cells (BHK and CH0, etc.), monkey-derived cells (COS and COS 3. COS7, CV1 and Ve1 o, etc.) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells and Nama wa), etc., and preferably L51. 78Y, COS 1 cells, COS 3 cells, COS 7 cells, cells derived from human diploid fibroblasts and myeloma cells

The selection of the transformed cells can be performed using a conventional cell selection technique by imparting one or more properties such as drug resistance, temperature sensitivity, auxotrophy or radiation mutability to the transformed cells. Depending on the purpose, the selection marker DNA used in the present invention is usually used in test research or in the industrial field. Such selectable marker DNA can be selected and used. For example, ampicillin (Amp) resistant DNA, tetracycline (Tc) resistant DNA, thymidine kinase (Tk) resistant DNA, 6-thioguanine (HGP RT) resistant DNA, neomycin (Neo) resistant DNA and hygromycin Drug-resistant DNA such as resistant DNA can be used.

 As a method for purifying and isolating the desired chimeric soluble Fas antigen from the culture supernatant of the transformed cells, the obtained culture is fractionated by filtration, centrifugation, or the like. It can be purified according to the method. That is, for example, methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, methods using molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and ion exchange chromatography. Hydroxyl abatite mouth chromatography A method using first-class charge, a method using specific affinity such as affinity chromatography, a method using difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, etc. A method utilizing isoelectric point such as point electrophoresis may be used.

 Here, the “substantially constituting amino acid sequence” means the extracellular region of the Fas antigen or the mouse IL-13 receptor; the extracellular region of the 3-subunit AIC2A in vivo. Means an amino acid sequence containing at least the minimum amino acid sequence necessary to maintain the function or biological activity, and deletion, modification or addition of N-terminal or C-terminal amino acids is allowed according to the purpose. To get

'Taste is o

 In addition, the monoclonal antibody of the present invention, specifically, an anti-Fas monoclonal antibody used for a solid-phased anti-Fas monoclonal antibody or a labeled anti-Fas monoclonal antibody, and a monoclonal antibody against the chimeric soluble Fas antigen. The null antibody can be produced by a known general method for producing a monoclonal antibody.

 For example, it can be produced from a hybridoma (fused cell) produced by so-called cell fusion. That is, a fused cell is prepared from antibody 產 living cells and myeloma cells (myeloma cells), and the hybridoma is cloned.

1 B Produce a monoclonal antibody with specific affinity to the isolated chimeric soluble Fas antigen, or a natural or artificially produced cell expressing the Fas antigen. Manufactured by selecting clones. For this operation, conventionally known means can be used except for using the aforementioned Fas antigen or Fas antigen-expressing cells as the immunizing antigen.

 Chimeric soluble Fas antigen as an immunizing antigen, natural or artificially-produced cells expressing the Fas antigen, and the like can be immunized with mice, rats, guinea pigs, hamsters, or egrets. Mammals, preferably mice, rats or hamsters. Immunization is carried out by subcutaneously, intramuscularly, intravenously, footpad II or intraperitoneally injecting or transplanting the immunogen into these mammals one to several times.

 Usually, animals are immunized 1 to 4 times about every 1 to 2 weeks after the first immunization, and finally immunized about 1 to 4 weeks later, and immunized about 3 to 5 days after the final immunization Antibody-producing cells are obtained from

 The preparation of a hybridoma that secretes a monoclonal antibody can be performed according to the method of Koehler and Mirushi Utain (Nature, 256, 495-497. 1975) and a modification method analogous thereto. That is, the monoclonal antibody in the present invention is preferably a mouse of the same species as an antibody-producing cell contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably spleen, obtained from the animal immunized as described above, Culturing a fusion cell (hypridoma) obtained by fusion with a mammal such as a rat, guinea pig, hamster, rabbit, human or the like, more preferably mouse, rat or human myeloma cell line (myeloma cell) Prepared by Cultivation can be performed in vitro or in vivo in a mouse, rat, guinea pig, hamster, or egret, etc., preferably in a mouse or rat, more preferably in ascites of a mouse, and the like. Can be obtained from ascites.

Examples of myeloma cells used for cell fusion include myeloma derived from mouse P3 / X63-AG8, P3 / NSI / l-Ag4-l (abbreviated as NS-1), P3 / X63-Ag8. UU SP2 / 0-Agl4, FO or BW5147, rat-derived myeloma 210RCY3_Ag.2.3., Human-derived myeloma U-266AR1, G 1500-6TG-A1-2, UC729-6, CE-AG, D1R11 or CEM-T15. .

 Screening of fused cell clones that produce monoclonal antibodies is performed by culturing the fused cells, for example, in a microtiter plate, and determining the reactivity of the culture supernatant, which shows growth, with the antigen, for example, RIA (radiation). Immunoassay) or enzyme immunoassay such as ELISA.

 Monoclonal antibody purification and isolation are performed by purifying blood or ascites containing the monoclonal antibody, which is obtained by the above-described method, by ion exchange chromatography (0 £ 8 £ or 0 £ 52, etc.) It can be performed by subjecting to affinity column chromatography such as a immunoglobulin column or a protein A column.

The monoclonal antibody in the present invention, specifically, an anti-Fas monoclonal antibody used for a solid-phased anti-Fas monoclonal antibody or a labeled anti-Fas monoclonal antibody, and a monoclonal antibody against the chimeric soluble Fas antigen are: Includes monoclonal antibodies belonging to any of the immunoglobulin classes of IgG, Ig. IgA, IgD and IgE. Preferably, the transformed cells or antibody-producing hybrids of the present invention are IgG or IgM. In culturing the doma, the transformed cells or fused cells are grown, maintained, and preserved in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culturing method. Any nutrient medium derived from a known nutrient medium or a known basal medium, such as those used to produce antibodies or antibodies It is possible to implement a medium Yore, Te. As a basic medium, for example, Ham 'F12 medium, low calcium medium such as MCDB 153 medium or low calcium MEM medium and MCDB 104 medium, MEM medium, D-MEM medium, RPMI 1640 medium, ASF104 medium, RD medium Medium, such as a high calcium medium or a Fisher's medium. The basic medium may be, for example, serum, hormone, cytokin, and / or An inorganic or organic substance can be contained.

 The immunoassay kit for a soluble Fas antigen and the immunoassay method for a soluble Fas antigen according to the present invention include an immunoassay kit for a soluble Fas antigen in a body fluid such as serum which has not yet been established. It provides the first immunological measurement method. Using the measurement kit and the measurement method, serum and other body fluids of patients suffering from autoimmune diseases such as rheumatoid (Rheumatoid Arthritis; RA) or SLE, which have been difficult to measure until now. It is possible to measure the amount of soluble Fas antigen easily, with high accuracy and high sensitivity. By measuring the soluble Fas antigen of the patient, it is considered that the dynamics of the Fas antigen such as the autoimmune disease is involved, foreseeing, diagnosing, establishing a treatment policy, and judging the effect of the treatment. The present measurement kit and measurement method are extremely useful clinically. Furthermore, the measurement kit and the measurement method of the present invention lack quantification and are unsuitable for processing a large amount of samples at a time. The stamp mouth method or a specific qualified person for using radioactive materials Is simpler, more accurate and more sensitive than radioimmunoassay (RIA), which can only be performed in a specific controlled facility. In addition, a large number of samples can be measured at one time. Is possible.

In addition, the method for producing a chimeric soluble Fas antigen of the present invention is a method for producing a soluble region of a transmembrane protein, such as a not-yet-established Fas antigen, as a soluble active substance in vitro in a simple and large amount. Is the first to offer. By using this production method, it is possible to produce the soluble region of the Fas antigen, which has been very difficult to obtain until now, as a soluble active substance simply and in large quantities at the in-vitro mouth. The chimeric soluble Fas antigen thus produced is required in a method for measuring a soluble Fas antigen such as a kit for immunological measurement of a soluble Fas antigen of the present invention and an immunological measurement method. Very useful as a purification standard (standard). Furthermore, the chimeric soluble Fas antigen is extremely useful not only as a standard substance but also as an immunizing antigen for producing a monoclonal antibody against a soluble region of the Fas antigen which is important as a ligand recognition site of the Fas antigen. It is. Hereinafter, the embodiments of the present invention will be described more specifically with reference to Examples, but it is needless to say that the present invention is not limited to the embodiments described below.

Example 1 Preparation of standard human soluble human Fas antigen

 The soluble human Fas antigen used as a standard was a chimeric soluble human Fas antigen and was prepared as follows.

 That is, the transformed cells JM109 prepared according to Example 7 described below (accession number FER BP-4436, deposited on October 6, 1999, at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry ) Using Gene Pulser (Bio-Rad Co., Ltd.) using Brasmid pMBl 8S / hFas.EXT-A 〖C2A.EXT and Brasmid PMAMneo (Clontech Co., Ltd.) Then, mouse lymphoma cells L5178Y (ATCC CRL1722) were co-transformed by electroporation. The cells were cultured in a medium containing the antibiotic G418, and a G418-resistant clone was selected to obtain a chimeric soluble human Fast antigen-expressing clone. The clone was mass-cultured using a 10% Poma serum-containing Fischer medium, and the culture supernatant was collected and concentrated, and then subjected to column chromatography using Q Sepharose (Pharmacia Co., Ltd.) as a standard substance. Were obtained chimeric soluble human Fas antigens.

Example 2 Production of anti-human Fas monoclonal antibody

 <2-1> Preparation of antibody cells

 A Ba1 bZc female mouse was immunized with human diploid male fibroblast FS-7 expressing human Fas antigen. The sensitized mouse was laparotomized, the spleen was excised, ground on a stainless steel mesh in serum-free RPMI 1640 medium, and the spleen cell solution was centrifuged (1500 rpm for 7 minutes). The centrifugation residue was collected and suspended in a bloodless RPMI 1640 culture solution. Furthermore, the cells were washed twice with a serum-free RPMI 1640 culture solution to obtain antibody-producing mouse spleen cells.

 <2-2> Preparation of mouse myeloma tsumugi vesicle

Mouse myeloma cells NS- 1 a (ATCC TIB18), at 37, 5% C0 ₂ under incubated in 1 0% FCS and 50 U / m 1 kanamycin-containing AS F- 1 04 medium (Ajinomoto (Ltd.)) did. <2-3> Preparation of anti-human Fa s monoclonal antibody-producing hybridomas Mouse-free myeloma cells NS-1 cells washed with serum-free RPMI 1640 medium and mouse spleen cells prepared in <2-1> were mixed. The RPMI 1640 solution was centrifuged (1000 rpm for 10 minutes), and the centrifuged residue was collected. Hertzenberg, L. (Herzenberg, L. Α ·) et al. (Selected Methods in Cellular Immunology. 351, 1980) ), And multiple colonies of hybridomas were obtained.

 To screen for hybridomas producing anti-human Fas monoclonal antibodies, flow cytometry was used to measure the reactivity of the culture supernatant of each hybridoma colony against human thymocytes expressing the Fas antigen. As a result, a hybridoma clone CH11 (I) producing an anti-human Fas monoclonal antibody was obtained.

 <2-4> Acquisition of anti-human F a s monoclonal antibody

 The hybridoma clone CHI1 (2X10, pcs.) Was cultured in serum-free ASF 104 medium (Ajinomoto Co., Inc.) at 37'C for 5 days, and the obtained culture supernatant was used for omega cell (Philtron Co., Ltd.). )). The supernatant was collected and supplied to a hydroxylapatite column (Asahi Optical Co., Ltd.). It was eluted with sodium phosphate (pH 7.4, 10-400 mM) using an FPLC system (Pharmacia 7 Chemical Co., Ltd.). The eluted fraction was collected, subjected to SDS-PAGE, and purified to a purity of 95% or more (see Journal of Experimental Medicine, Vol. 16, pp. 1747-1756, 1989). Antibodies can be purchased from the Institute of Medical Biology, Inc.

 Further, other anti-human Fas monoclonal antibodies were prepared as follows.

<2-5> Preparation of immunizing antigen

A fragment containing the human Fas antigen cDNA obtained by digesting the plasmid pF58 containing the cDNA encoding the human Fas antigen with XhoI was used as an expression vector pEF—BOS (Nucleic acid research, 18, p. 5322, 1990) using a BstXI adapter to obtain an expression plasmid pEFF58. With V sp I digested was 2 5 GZml of Peff 5 8 Fragment 2-5 / was digested with the E c oR I £ g / ml OpMAn eo ( Clontech (Ltd.)) fragment, 1 1 0 ⁷ (0.8 ml) of mouse T lymphoma WR19L cells (ATCC TIB52) were co-transformed by electoporation.

 The cells were cultured in a medium containing the antibiotic G418 to select G418 resistant clones. The obtained G418 resistant clone was analyzed by a flow cytometer and an ultradilution method to clone a transformant cell WR19L12a overexpressing human Fas antigen (CELL, Vol. 66, 233-243, 1991).

 WR19L12a cells were homogenized and centrifuged to obtain a plasma membrane surface, which was used as an immunizing antigen.

 <2-6> Preparation of antibody-producing cells

 The B1bZc mouse was immunized with the plasma membrane fraction of WR19L12a, a human Fast antigen-overexpressing transformed cell, and antibody-producing mouse spleen cells were obtained in the same manner as described above.

 <2-7> Preparation of anti-human F s 乇 noclonal antibody 產 live hybridoma

 Using the above mouse myeloma NS-1 and the obtained mouse spleen cells, a plurality of hybridoma clones were obtained in the same manner as described above, and seven anti-human Fas monoclonal antibody-producing hybridoma clones, ZB 4 (IgG), UB2 (IgG), VB3 (IgG), CBE (IgG), WB3 (IgG), AX6 (IgM) and JAE (Ig) .

2— 8> Obtaining anti-human F a s monoclonal antibody

 In the same manner as described above, seven types of anti-human Fas monoclonal antibodies were purified and obtained from each of the seven obtained hybridoma clones (International Immunology. Vol. 6, No. 12, pp. 1849-1856, 1994 ).

 In addition, these anti-human F a s monoclonal antibodies can be purchased from Institute for Medical Biology, Inc.

Example 3 Immobilization of anti-human F a s monoclonal antibody on insoluble support (Preparation of microplate-type reagent)

Anti-Fas monoclones obtained as described above from Hypri-Doma clone CBE The IgG fraction of the lonal antibody was dissolved in a 0.1 M phosphate buffer (pH 7.4, containing 0.1% sodium azide) to adjust the concentration to 0.005 mg / ml. This solution was added to each well of a 96-well microplate (Maxi Soap, manufactured by Nunc Corporation) in a quantity of 1001 and allowed to stand at 4'C for about 18 hours to bind the antibody to the microplate. .

After binding the antibody, remove the monoclonal antibody solution in the gel, and add 300% / zl of phosphate buffered saline (PBS) containing 1% serum albumin (BSA) and 5% sucrose to each gel. The reaction was allowed to stand at 4 ^eC for 24 hours to allow unreacted portions of each well of the microplate to be blocked. This blocking is performed so that the labeled antibody or the like added during the measurement of the sample is not adsorbed on the wall surface of the microplate, so that the quantitativeness of the measurement system is not impaired. After the blocking, the blocking solution in the well was removed and completely air-dried at room temperature to obtain a microplate type reagent (an immobilized anti-Fas monoclonal antibody).

Example 4 Preparation of labeled anti-Fas monoclonal antibody

 From the hybridoma clone VB3 as described above, according to the method described in the journal 'Ob' Histochemistry and Cytochemistry (J. Histochem. Cytochem.), Vol. 22, pp. 1084 (1974). Anti-Fas monoclonal antibody I 80 fractions 5111 £ were combined with POD (Horse radish peroxidase. Sigma) by the periodate method to obtain a peroxidase-labeled anti-Fas monoclonal antibody (labeled A second anti-Fas monoclonal antibody). Example 5 Measurement of soluble Fas antigen (Part 1)

 A kit for measuring soluble Fas antigen was prepared by combining the microplate-type immobilized anti-human Fas monoclonal antibody and the peroxidase-labeled anti-Fas monoclonal antibody prepared in Examples 3 and 4, respectively. The standard substance chimeric soluble human Fas antigen was measured to prepare a calibration curve, and the soluble Fas antigen in the human serum sample was actually measured.

 <5-1> Preparation of serum sample and standard soluble Fs antigen

Healthy individuals (200), SLE patients (50) and RA patients (50) Serum obtained from slightly blood by a conventional method was diluted 50-fold with PBS to prepare a sample. The standard chimeric soluble human Fas antigen prepared in Example 1 was similarly diluted 50-fold with PBS to obtain a standard solution. When the amount of the soluble Fas antigen contained in the sample was large and exceeded the detection limit, the sample was further diluted with PBS so as to be within the measurable range. In this case, the concentration of the soluble Fas antigen in the sample was determined by multiplying the quantitative value of the diluted sample by a dilution factor.

 <5-2> Antigen-antibody reaction between immobilized monoclonal antibody (microplate type reagent) and soluble F ss antigen in sample and standard solution

 The microplate type reagent (immobilized monoclonal antibody) prepared in Example 3

The sample or standard solution prepared in <5-1> was dispensed at a concentration of 100 l Z ゥ ヱ. In addition, a bottle in which PBS alone was dispensed in 1001 wells was blinded. The reaction was allowed to stand at room temperature for 1 hour to react. After the completion of the reaction, each well was washed three times with 5% PBS, and the PBS was removed well.

 <5-3> Binding of peroxidase-labeled anti-Fas monoclonal antibody to conjugate of immobilized monoclonal antibody (microplate type reagent) and soluble Fs antigen in sample or standard solution

 The peroxidase-labeled anti-Fas monoclonal antibody prepared in Example 4 was concentrated at a concentration of 250 to 1,000 times with 1 OmM sodium phosphate buffer (pH 8.0) supplemented with 0.1% BSA and 0.15 M NaCl. Diluted. The diluted solution was dispensed at a concentration of 100 ゥ Z ゥ l into a microplate that had been subjected to an antigen-antibody reaction with 一> 2>, and allowed to stand for 1 hour to react. After the completion of the reaction, each well was washed three times with PBS, and the buffer was removed well.

 <5— 4> Color reaction

Tetramethylbenzidine dihydrochloride (Sigma) was dissolved in 1 OmM citrate buffer (pH 6.8) to prepare a 1.6 mM tetramethylbenzidine dihydrochloride solution. Hydrogen peroxide was diluted with 1 OmM citrate buffer (pH 6.8) to prepare a 1 OmM hydrogen peroxide solution. Equal amounts of the 1.6 mM tetramethylbenzidine dihydrochloride solution and 1 OmM hydrogen peroxide solution were mixed to obtain an enzyme substrate solution. The substrate solution is dispensed at 100 ° C / degree S in microplates reacted with peroxidase-labeled anti-Fas monoclonal antibody in <5-3>, and left at room temperature for 10 to 20 minutes And reacted. Next, 1.5N phosphoric acid was dispensed to each gel at a concentration of 100 w 1 Z gel, and the enzyme activity of peroxidase was stopped by lowering the pH.

 The degree of color development generated in each microplate plate was detected by measuring the absorbance at a wavelength of 450 nm using an absorptiometer (PR-A4, manufactured by BANDOSHI Co., Ltd.). The concentration of the soluble Fas antigen in the sample was derived from a calibration curve prepared by performing measurement as described above using a standard solution based on the detected absorbance. Table 1 shows the measurement results using the standard solution, and Fig. 1 shows a calibration curve based on the measurement results. Table 2 shows the measurement results of soluble Fs antigen in serum samples of healthy subjects, SLE patients, and RA patients derived based on the calibration di. Table 1 Standard soluble Fs antigen concentration (ng / ml) Absorbance (A450nm)

0.0 (blank) 0.076

1. 0 0. 300

2. 0 0.51 2

4.0 0.830

8.0 1.256

1 6.0 1. & 98

32. 0 2.004 Table 2

Example 6 Measurement of soluble Fas antigen (Part 2)

 <6-1> Immobilization of anti-human F a s monoclonal antibody on insoluble support (preparation of microplate type reagent)

 Anti-Fas monoclonal antibody IgG fraction obtained from Hypri-Doma clone CBE as described in Example 2-8> was added to 0.1 M phosphate buffer (pH 7.4, containing 0.1% sodium azide). And adjusted to a concentration of 0.01 mg / ml. Add 100 μl of this solution to each well of a 96-well microplate (manufactured by Nunc Co., Ltd.) and let stand for about 18 hours at 4 to bind the antibody to the microplate. I let it.

 After binding the antibody, remove the monoclonal antibodies in the wells and add 300% phosphate buffered saline (PBS) containing 1% serum albumin (BSA) and 10% sucrose to each well. The mixture was allowed to stand at 37 for 2 hours to react, and the unreacted portion of each well of the microplate was blocked. This blocking is to prevent the labeled antibody or the like added during the measurement of the sample from being adsorbed on the wall surface of the microphone opening plate, so that the quantitativeness of the measurement system is not impaired. After the blocking, the blocking solution in the well was removed and completely air-dried at room temperature to obtain a microplate type reagent (immobilized anti-Fas monoclonal antibody).

<6 -2> Production of a recognized anti-Fas monoclonal antibody

The IgG fraction of the anti-Fas monoclonal antibody obtained from the hybridoma clone VB3 as described in Example <2-8> was passed through 0.1 l acetate buffer (pH 4.2). After the analysis, the protein concentration was adjusted to 5 mg / ml, 3% of the total protein amount of vepsin was added, and the mixture was stirred at 37 overnight to carry out the reaction. The reaction was stopped by adding 2 M Tris-HCl buffer (pH 8.0) all at once, and then ultragel equilibrated with 0.1 M phosphate buffer (pH 6.5) containing 0.2 M sodium chloride. Gel filtration was performed using an Ac A44 (manufactured by LKB) column (2.0 × 60 cm) to obtain _2.5 mg of F (ab ′) ₂ fraction.

 Succimidyl 4- (N-maleimide-methyl) -cyclohexane-11-carboxylate (Succimidyl 4- (N-raaleiraidomethyl) -cyclohexan-l-carboxylate, Ziehen Chemical Co., d.) The POD (Horseradish peroxidase, Sigma) was obtained according to the hinge method (J. Immunoassay, vol. 4, p. 209, 1983) using the mouse anti-Fas monoclonal antibody obtained above. conjugated to gG / Fab 'via SH group to obtain a peroxidase-labeled anti-Fas monoclonal antibody (labeled second anti-Fas monoclonal antibody)

7

 <6-3> Measurement of soluble Fa s antigen

 The microplate-immobilized anti-human Fas monoclonal antibody and the peroxidase-labeled anti-Fas monoclonal antibody prepared in <6-1> and <6-2> above were combined to form a kit for measuring soluble Fas antigen. Using the kit, the standard substance chimeric soluble Fas antigen was measured to prepare a standard, and the soluble Fas antigen in the human blood sample was actually measured.

 A sample was prepared from serum obtained from peripheral blood of each of healthy subjects (200), SLE patients (50) and RA patients (50) in the same manner as in Example 5 A standard solution was prepared from the standard chimeric soluble human Fas antigen prepared in Example 1. Using these specimens or standard solutions, an anti-progenitor antibody reaction was performed with the microplate-type reagent (immobilized monoclonal antibody) prepared in <6-1> in the same manner as in Example 5 <5-2>. . Next, the peroxidase-labeled anti-Fas monoclonal antibody prepared in <6_2> was bound to the conjugate of the microplate-type reagent (the immobilized monoclonal antibody) and the soluble Fas antigen in the sample or standard solution.

2 g The color reaction was performed as follows. Orto Phenylene Diamine (Sigma)

Was dissolved in a 0.1 M sodium citrate buffer (pH 5.1) to a concentration of lmg / ml, and 1001 / well was added to a microplate reacted with a peroxidase-labeled anti-Fas monoclonal antibody. And allowed to stand at room temperature for 10 to 20 minutes to react. Then, 2N sulfuric acid was dispensed at a concentration of 100/1 / perol to each pellet, and the enzyme activity of peroxidase was stopped by lowering the pH. The degree of color development in each well of the microplate was detected by measuring the absorbance at a wavelength of 492 nm using an absorbance meter (manufactured by Tosoichi Co., Ltd., MPR-A4). The concentration of the soluble F ss antigen in the sample was derived from a calibration curve prepared based on the detected absorbance by performing measurement as described above using a standard solution. Table 3 shows the results of measurement using the standard solution, and FIG. 2 shows a calibration curve based on the measurement results. Table 4 shows the measurement results of soluble Fs antigen in serum samples of healthy subjects, 51 ^ £ patients and 118 patients derived based on the calibration curve.

 '' Table 3 Standard soluble Fs antigen concentration (ng / ml) Absorbance (A492mn)

0 (blank) 0, 1 22

 1 0 0, 300

 20 0.5 1 2

 40 0. 830

 80 1.256

 1 60 1. 6 98

320 2.004 Table 4

 As shown in Tables 2 and 4, soluble Fas antigen was detected in the sera of healthy subjects, SLE patients and RA patients, and significantly higher concentrations of soluble Fa antigens were detected in SLE and RA patients than in healthy subjects. It can be seen that s antigen was detected, and particularly in SLE patients, a remarkably high concentration of soluble Fa s antigen was detected.

 Therefore, the concentration of soluble Fas antigen in body fluids of healthy persons and patients can be easily measured by implementing the kit for immunological measurement of soluble Fas antigen and the immunological measuring method of the present invention at clinical sites. be able to. In particular, to predict, discover, diagnose, diagnose treatment, establish treatment policies, and determine the efficacy of treatment for various diseases in which the dynamics of Fas antigens such as autoimmune diseases in 31 ^ 8 patients and 108 patients are considered to be involved. It is not possible to do it quickly.

Example 7 Preparation of anti-Fas monoclonal antibody labeled with biotin

 In place of the peroxidase-labeled anti-Fas monoclonal antibody prepared in <6-2> of Example 4 or Example 6, an anti-Fas monoclonal antibody prepared with piotin prepared as described below was used. The soluble Fas antigen can be measured in the same manner as in Examples 5 and 6.

The IgG fraction of the anti-Fas monoclonal antibody obtained from the hybridoma clone VB3 as described in Example 2-8> was dissolved in 0.11 ^ 崁 -acid buffer (158.5) and the IgG concentration was Was adjusted to 5 mgZm1. NHS-LC-B I OT IN (manufactured by PI ERCE) similarly prepared by dissolving the IgG solution in 0. IMiJt acid buffer (pH 8.5) to prepare 25 mg nom 1 ) To the IgG 1 0 mg was added, and the mixture was stirred at room temperature for 4 hours using a stirrer to prepare a biotin-labeled anti-Fas monoclonal antibody.

 When the biotin-labeled anti-Fas monoclonal antibody obtained in this manner is used, the enzyme (peroxidase, galactosidase, etc.) labeled with avidin is further reacted as described above, and the immobilized anti-Fas monoclonal antibody is used. Form a complex of mono-soluble anti-Fas anti-Piotin-labeled anti-Fas monoclonal antibody and avidin-labeled enzyme, and measure the color of the chromogenic substrate in the same manner as in Examples 5 and 6 to determine whether it is in the sample or in the standard solution. The concentration of soluble Fas antigen was measured.

 Even when a combination of a biotin-labeled anti-Fas monoclonal antibody and an avidin-labeled enzyme is used, the same high sensitivity and solubility as in the case of using a normal enzyme-labeled monoclonal antibody as in Example 5 or 6. Example 8 Preparation of Transformed Cell JM1 09 (International Deposit No. FERM ΒΡ-4436)

 The transformed cells JM109 used in the preparation of the standard chimeric soluble human Fas antigen in Example 1 were prepared as follows.

 <8-1> Acquisition of cDNA encoding full-length human Fas (hFas)

<8-1-1 A> Cell line preparation

 The cell line used in this example was prepared in advance.

 • Human T tumor cell line KT3 (available from Dr. Shimizu, Kanazawa Medical University): Contains 10% fetal calf serum (FCS) and 5 ng / 1 human recombinant IL-16 (Ajinomoto) Cultured in RPMI 1640 medium.

 'Monkey cell line COS 7 (ATCC CRL 1651): cultured in DMEM medium containing 10% FCS.

 <8— 1— B> Preparation of antibody

 The mouse anti-human Fas antibody CH11 (IgM) used in this example was prepared in advance in the same manner as in Example 2 (Yonehara et al., J. Exp. Med .. Vol. 169, 1747- 1756 p. 1989) 0

<8-1 -0 Brassid construction A foreign cDNA cloning vector (Blasmid) used in this example was constructed in advance.

 • Plasmid pCEV4 (Science, 247: 324-327; see 1990, Itou et al.): Plasmid pcDSRc Mol. Cell Biol., 2: 161, 1982, Okayama et al.

Mol. Cell Biol., Vol. 3, pp. 280, 1983, Okayama et al. And MoL Cell Biol., Vol. 8, pp. 466-472. 1988, Takebe et al. replication origin DNA (Py0 ri) and plasmid CDM8 (Proc. Natl. Acad. Sci. USA. 84, 3365-3369, 1987) Stuffer with two Bst XI cleavage sites, taken as XbaI cleavage fragments from Brian Seed et al. And Brian Seed et al. And Nature, Vol. 329.840-842. 1987. Brian Seed et al. ) DNA was inserted into the NdeI cleavage site and PstI cleavage site of pcDSRa, and the Notl cleavage site in the stuffer (XbaI cleavage fragment) was removed by filling in, and the NotI linker was removed. The pcDSRa C1A

The plasmid PCEV4 was constructed by inserting it into the I cleavage site.

 • Brasmid pME18S (available from Dr. Kazuo Maruyama, The University of Tokyo): A cDNA cloning vector suitable for expression in animal cells constructed using PCEV4 as the basic skeleton. The replication origin sequence of pUC, SV40 16S intron sequence, p

01 y A signal sequence and ρ 01 y A signal sequence immediately before the termination of each frame (Experimental Medicine “Genetic Engineering Handbook J (separate volume), pp. 101-107. 1992, see Maruyama Kazuo et al.) .

 Restriction enzyme maps of Brasmid PCEV4 and pME18S are shown in FIGS. 3 and 4, respectively.

 <8-1-1 D> Preparation of cDNA library derived from KT3 human T tumor cells

Total RNA was prepared from KT3 by the guanidine isothionate / acid phenol method (Anal. Biochem., Vol. 162, pp. 156-159, 1987, Chomczynski and Sacchi et al.), And poly (A ) RNA was selected by o 1 igo (d T) cellulose column chromatography. Random hexamer oligonucleotide (pdN6) ( Using M-MLV RNAase H "reverse transcriptase as a primer with BRL (manufactured by) and oligo (dT) (BRL (manufactured by)), double-stranded cDNA was synthesized from the RNA (Cell, No. 1). 61, pp. 341-350, 1990; see Fukunaga et al.) A pre-synthesized Bst XI non-palindromic adapter was ligated, cDNAs longer than 2 kb were separated by agarose gel electrophoresis, and these were separated into Bs 1 The vector was ligated to the vector fragment by electroporation (see Nucl. Acids Res., Vol. 16, pp. 6127-6145. 1988, Dower et al.). Each of these cells was transformed into a cDNA library derived from human T tumor cells KT3.

 The DNA sequence of the B st X I non-palindromic adapter used is shown in SEQ ID NO: 1.

 <8-1 -E> hF a s c DNA fragment acquisition

 The following operation was performed in the same manner as in the method of Brian Seed et al. (Pro Nat 1. Acad. Sci. USA. 84, 3365-3369, 1987).

Bacteriological 6 cm dish (Falconl007) containing 10 // g goat anti-mouse IgM antibody (Cappel, Inc.) 5 OmM Tris— HC1 (3 ml; H9.5) at room temperature 90 The unreacted antibody was removed by washing the panning dish three times with 0.15 M NaC 1 to remove the unreacted antibody, then adding 0.5 mM EDTA and 0.02% The cells were blocked by overnight incubation using PBS (phosphate-buffered saline) (3 m) containing NaN ₃ and 5% FCS.

When COS 7 seeded on 108 6 cm dishes (Falconl007) grew to 50%, spheroplast fusion; Mol. Cell. Biol., Vol. 1, pp. 743-752, 1981, Sandi-Goldrin et al.) Transformed the previously prepared cDNA library into COS7. After 72 h, 0. 5 mM of EDTA及beauty 0.02% of NaN ₃ PBS containing (PBSZEDTA / NaN ₃₎ was added, the cells were the剝from dishes by incubation for 30 minutes at 3 7 ^e C. The collected cells were collected, centrifuged, and 10 / g / ml mouse anti-human Fas antibody CHI 1 ( Ig) (see J. Exp. Med .. 169, 1747-1756, 1989, Yonehara et al.) In ice-cold PBS / EDTA NaN ₃ (9 ml). After incubation on ice for 60 minutes, PBS _/ EDTAZNaN ₃ with钿胞l: 2 diluted and overlaid 2% Fuikoru (Ficoll) 400, and centrifuged at 1000 r pm. Contains 5 to 3 viable cells collected in the upper layer of Ficoll? 83 / £ 0 chohachi / ^^ 31 ^ ₃

(27 ml), and the precipitate was removed by filtration with a nylon mesh (pore size: 10 Owin).

The filtered cells were plated on the previously prepared banning dish (54). Leave at room temperature for 2 to 3 hours to allow the cells to adhere to the dish, and then wash gently three times with PBS / EDTA / NaN ₃ (2 ml) containing 5% FCS to remove cells that do not bind to the dish . According to the method of Hirt et al., Plasmid DNA was prepared from the COS cells bound to the dish. That is, a 0.6% 303 solution (0.4 ml) containing 1 OmI [ED chohachi] was added to each dish and left at room temperature for 20 minutes. The lysate was collected in a microtube, NaCl was added to a concentration of 1M, and the tube was allowed to stand on ice for 5 hours or more. Next, the mixture was centrifuged at 13000 rpm for 5 minutes, and the supernatant was extracted with phenol-chloroform. Brassamide DNA was recovered by ethanol precipitation. Recovery Burasumi de DNA in the E. C o 1 i WM 1 1 0 0 was transformed to give about 3. 2 X 1 0 ⁵ colonies, after selecting the transformants conversion cells analyzed colonies, staple port Transformed into COS 7 by plast fusion

(48 dishes). The same operation was further repeated twice to obtain 14 plasmid clones containing the hF s cDNA fragment (pF1-pF14). These plasmid clones were cut with the restriction enzyme XhoI and clones containing the same 3. Okb insertion sequence (PF1, 2, 5, and 11) and clones containing the same 1.5 kb insertion sequence. (PF 3, 4, 6, 7, and 9). After transforming the pF1 and pF3 plasmid clones into COS7, these clones should be cDNA fragments encoding the Fas antigen by flow cytometry analysis using an anti-Fas antibody. It was confirmed.

<8-1 -F> Obtain full-length hF as cDNA The colony hybridization method using the 0.5 kb Xhol-BamHI cleavage fragment at the 5 'end of pF3 obtained in <8-1-1 E> Ten cDNA clones were obtained from the cDNA library created in D>. Restriction enzyme mapping confirmed that these clones contained a 1.8-2.6 kb insert with the same restriction map. According to various analyses, the longest clone, pF58, consists of 2534 bp, starting from the 3 'terminal polyadenylation signal sequence (ATT AAA), the translation initiation codon located at bases 195 to 197. It has an open reading frame of bρ and a translation stop codon (TAG) located at bases 1200 to 1202, confirming that clone pF58 has full-length hF asc DNA. . A 2555 bp DNA containing a full-length Fas antigen cDNA extracted as a XhoI digestion fragment from pF58 green was incorporated into a mammalian expression plasmid PCEV4 using a Bst XI adapter to construct a plasmid pCEV4ZhFas.

 The cDNA sequence encoding the full-length hFas and the corresponding amino acid sequence are shown in SEQ ID NOs: 2 and 3, respectively.

8—2> cDNA encoding the extracellular domain of hFas (hFas · EXT)

 Get

 The pCEV4 hFas constructed in <8-1-1 F> was digested with XhoI, and the full-length hFas cDNA was extracted as an XhoI digestion fragment (2555 bp). Also, pME18S was digested with XhoI, and a portion of the stuffer was removed as an XhoI digestion fragment. The pME18S fragment was treated with alkaline phosphatase (BAP) (E. coli C75; Takara Shuzo Co., Ltd.) to remove the phosphate group at the 5 'end of the cut surface. The plasmid pME18SZhFas was constructed by ligating the full-length hFascDNA to the XhoI-cleaved fragment (2555 bp) and the BAP-treated pME18S fragment using a DNA ligation kit (Takara Shuzo).

pME 18 SZhF as is digested with Bg1 II and PstI, and transmembrane of hFas The cDNA region corresponding to the cloning region and the subsequent intracellular region was removed. The blunt ends were then blunted using the DNA blunting kit (Takara Shuzo), the pME18S fragment was self-ligated, and the plasmid pME18 containing only the cDNA encoding hFas · EXT was introduced. S / hFa s · EXT was constructed.

 Brasmid pME18S / hFas ■ EXT construction process is shown in Fig. 5, and the cDNA sequence encoding the extracellular region of hFas (hFas · EXT) and the corresponding amino acid sequence are shown in SEQ ID NOs: 4 and 4, respectively. 5

 <8-3> mouse IL-1 3 receptor / 3 subunit AI C2A (AI C2A)

 Of cDNA encoding

8— 3— A> Preparation of cell line

 The cell line used in this example was prepared in advance.

 • Mouse obese cell line MC / 9 (ATCC CL 8306): Proliferated in RPMI 1640 medium containing 100 U / m1 recombinant mouse IL-13 and 10% FCS.

 • Monkey cell line COS 7 (ATCC CRL 1651): Propagated in DMEM medium containing 10% FCS.

 <8-3-B> Preparation of antibody

 The mouse anti-AIC2 antibody (IgM) used in this example was prepared in advance according to the report of Yonehara et al. (Int. Immunol. Vol. 2, No. 2, pp. 143-150, 1990).

 <8— 3— C> Construction of Plasmid

 We used the brass imide PCEV4 constructed in 8-11.

 8— 3— D> Construction of a cDNA library derived from mouse fertilizer cells MCZ 9

A double-stranded cDNA was synthesized from p01y (A) RNA selected from MCZ9 in the same manner as described in <8-1-1 D>, and ligated to Bst XI non-palindromic adapter. CDNAs longer than 1.5 kb were separated by electrophoresis and ligated to the BstXI cut pCEV4 vector fragment. The vector was transformed into E. coli DH5α by the electroporation method to prepare a cDNA library derived from mouse mast cell MCZ9 (Science, Vol. 247, 324-327, 1990, see Itto et al.). ). <8-3-E> Acquisition of AIC 2 A cDNA fragment

 A panning dish was prepared by coating a goat anti-rat IgM antibody (Cappel) as a secondary antibody in the same manner as described in <8-11). By using rat anti-Aic 2 antibody (IgM) as the secondary antibody, 24 plasmid clones transformed with COS 7 were obtained, and each clone was analyzed. After confirming that it had a 6 kb AI C2A cDNA fragment (insertion sequence), a brassmid clone pAI C2-2 was selected for use as a probe for the next step (Science. Vol. 247, pp. 324-327). , 1990, see Itou et al.).

8-1 3— F> Acquisition of full length AIC2Ac DNA

 A cDNA library prepared in 8-3-D> by the colony hybridization method using pAIC2-2 as a probe in the same manner as described in <8-1-1F>. From this, 18 cDNA clones were obtained, and a cDNA clone pAIC2-26 having full-length AIC2A cDNA was obtained (GenBank accession number: M29855). According to various analyses, pAIC2-26 was composed of 3351 bp and had an open reading frame encoding 878 amino acid residues. The oven reading frame was a signal peptide consisting of 22 amino acid residues. And a mature protein consisting of 856 amino acid residues, consisting of an extracellular region consisting of 417 amino acid residues, a transmembrane region consisting of 26 amino acid residues, and 413 amino acid residues. It was confirmed that the area consisted of the following areas.

DNA containing the full-length AIC2AcDNA extracted from the PAI C2--26 clone as an XhoI-cleaved fragment was incorporated into the mammalian expression plasmid pCEV4 using the BstXI adapter to construct the plasmid PCEV4ZA IC2A to construct the full-length AIC2. The cDNA sequence encoding A, the corresponding amino acid sequence, the cDNA sequence encoding the extracellular region of AIC2A (AIC2A • EXT), and the corresponding amino acid sequence are shown in SEQ ID NOs: 6 to 9, respectively. <8-4> hF as ■ Acquisition of cDNA consisting of cDNA encoding EXT and cDNA encoding extracellular region of AIC2A (AI C2A * EXT)

 The plasmid pME18SZhFasEXT constructed in <8-2> was digested with NotI, linearized, and the blunt end was blunted using a DNA blunt kit, and the phosphate group at the 5 'end of the cut surface was digested. Was treated with BAP to remove.

 By digesting the plasmid p CEV4ZA IC2A constructed in <8-3-1-F> with Dra [II, the cDNA encoding AIC2A • EXT was extracted, and the DNA was digested with a DNA terminal blunting kit. The cut ends of the fragments were blunted.

 Using a DNA ligation kit, the DralU-cleaved AIC 2A • EXTC DNA fragment was introduced into the NotI-cleaved pME 18 S / hFas • EXT brassmid fragment previously prepared, and the Brassmid pME 18 S / hF as · EXT-A IC 2 A · EXT was constructed.

 Figure 6 shows the construction process of pME 18 S / hF as-EXT-A IC 2AEXT, and the cDNA sequence encoding hF as EXT-A IC 2AEXT and the corresponding amino acid sequence. These are shown in SEQ ID NOs: 10 and 11, respectively.

 <8-5> Transformation of E. Co 1 i cell line JM109 with plasmid pME 18 S / hFas · EXT-AIC 2A · EXT

 Plasmid pME18S / hFas-EXT-AIC2A.EXT constructed in <8-4> was transformed into E. coli cell line JM109, and colonies were analyzed to select transformed cells. The transformed E. coli cell strain JM109 was deposited internationally with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry (Accession number: FERM BP-4436, date of deposit: October 6, 1993). ).

 <8-6> Expression, purification and analysis of chimeric soluble human Fas antigen (hFas-AI C2A)

Plasmid pME 18 S / hF as · EXT-A IC 2 A · EXT was extracted from the transformed cells JM109 by a conventional method, and electroporation was performed (Nucl. Acids Res., Vol. 16, pp. 6127-6145. 1988). Year. See Dower et al.) Was transformed.

 After the transformed COS 7 is cultured for 45 hours in a 0-1 ^ 1 ^ medium containing 10% 55, a serum-free culture obtained by culturing for 12 hours in a bloodless D-MEM medium is obtained. After centrifugation, the centrifuged supernatant was collected. The supernatant was centrifuged to purify a chimeric soluble protein comprising the hF s extracellular region and the AIC 2A extracellular region by column chromatography using Q Sepharose (Pharmacia Ryosha).

 Analysis of the purified soluble hFas-AIC2A protein by SDS-polyacrylamide gel electrophoresis confirmed that the obtained soluble protein was soluble hFas-A1C2A protein. Was done.

 The results are shown in FIG.

 In order to further confirm the properties of the purified soluble hFas-AIC2A protein, the soluble hFas-AIC2A protein was tested for cell death due to apoptosis of Fas antigen-expressing cells induced by anti-human Fas antibody. The effect of the suppression was investigated.

 25 1 mouse anti-human Fas antibody CH11 (10% 3/3 content scale? 1 ^ 11 640 in culture solution, 120 ng / m1) and 10% prepared in <8-1-1B> Each of the aforementioned oval soluble hF as—AIC 2A diluted to a concentration of 1/8, 1/1/6, 1/32, 1/64 and 1 128 with FCS-containing RPMI 1640 culture medium was added. The mixed solution with (1) was added to a 96-well micro tie evening plate (3 wells for each sample), and incubated at 37 for 30 minutes. Next, human-derived HP B-ALL cells confirmed to be sensitive to the mouse anti-human Fas antibody were added to each cell at a concentration of 1 × 10 * cell / ffell, and cultured for 20 hours. . After culturing, the cells were subjected to MTT assay (Immunology Letters, Vol. 19, pp. 261-268, 1988), and the viable cell ratio (%, average of three measured values) was measured using a microplate reader (0. D. 595-655). ) Was measured.

Further, soluble AIC 2A obtained by using pME18S / AIC2A · EXTT reconstructed from PCEV4ZA IC2A constructed in 8-3-F> was converted to RPMI1640 containing 1056FCS. Control experiments were performed in the same manner as described above, using solutions diluted to 1/8, 1/16, 1/32, 1Z64 and 1/128 with the culture solution. Cell death due to apoptosis of the Fas antigen-expressing cells induced by the anti-human Fas antibody was significantly suppressed in a highly correlated manner by the addition of the soluble hFas-AIC2A protein obtained in this example. Therefore, it was confirmed that the chimeric soluble protein obtained in this example had an intact function as a soluble human Fas antigen.

 The results are shown in FIG.

Example 9 Production of soluble mouse Fa s antigen (mFa s)

 <9-1> Acquisition of cDNA encoding full-length mFas

Preparation of cell lines

 The cell strain used in this example was prepared in advance.

 'Mouse macrophage BAM3 (see Cell. Physiol., Vol. 114, p.291, 1983, Ooki et al.): Cultured in D-MEM (Nissui Pharmaceutical Co., Ltd.) containing 10% FCS.

 <9-1 -B> Creation of a cDNA library derived from mouse macrophage BAM3

Total RNA from BAM3 treated with LPS (lipolivosaccharide) by the guanidinoisothiocyanate phenol method (Anal. Biochem., Vol. 162, pp. 6-159; see 1987, Chomczynski and Sacchi et al.). Was prepared, and the poly (A) RNA was selected by oligo (dT) cellulose column chromatography. A double-stranded cDNA was synthesized from the RNA using oligo (dT) as a primer (Nature, Vol. 319, p. 415, 1986, Nagayu et al.). The double-stranded cDNA was methylated with EcoR I methylase, ligated with EcoR I linker, digested with EcoR I, and subjected to agarose gel electrophoresis (low temperature gel) to obtain a cDNA longer than 70 Obp. These were ligated to an Ig 11 vector (Stratagene (manufactured by Stratagene)). , Respectively which the vector into E. C 01 i Y 1090 by electroporation volley Chillon method was transformed to prepare a mouse macrophage BAM3 derived cDNA library ^(3. 2 X 1 0 β plaques).

 <9-1 -C> Acquisition of full length mF a s c DNA

Digest the hFa sc DNA obtained in <8-1-1 F> with Xho I and BamHI. And the X ho I one B a mH I cut Colony High Priestess die See Chillon method using as a Burobu fragment obtained hF the as in 6. 5 X 1 0 ⁵ plaques the subscription twelve Ngushi five Positive clones (λΜΡ1 to 5) were obtained. The longest fragment of the 0.5 to 1.5 kb cDNA fragment obtained by digesting the cDNA obtained from the clone with EcoRI was used as the brassmid pB1uescript KS (+) ( The plasmid was subcloned into Stratagene (manufactured by Stratagene) to construct a plasmid pMF1.

 According to pMF1 restriction enzyme mapping and DNA sequence analysis, clone pMFI was composed of 1479 bp, 3 ′ terminal polyadenylation signal sequence (ATT AAA), translation initiation codon (ATG ) And a translation stop codon (TAG) located at bases 1031 to 1033, confirming that clone pMF1 has full-length mFasc DNA. J. I Set unol., 148: 1274-1279; 1992, see Fukunaga et al.).

 The cDNA sequence encoding full-length mFas and the corresponding amino acid sequence are shown in SEQ ID NOs: 12 and 13, respectively.

 <9-1-2> cDNA encoding the extracellular region of mFas (mFas · EXT)

 Get

 The pME18S constructed in <8-1-1C> was digested with EcoRI and XbaI to prepare a pME18S fragment. On the other hand, the pMF1 constructed at 9> 11C> was digested with EcoRI and F0kI to obtain a cDNA fragment encoding the extracellular region of mFas. To ligate this cDNA fragment to the pME18S fragment prepared above, a linker (ligated) DNA was prepared using a DNA synthesizer (DNA synthesizer). Using a DNA ligation kit, the mFas cDNA fragment and the linker DNA sequence were ligated to the pME18S fragment to construct plasmid pME18S / mFas · EXT.

The used phospho-DNA sequence is shown in SEQ ID NOs: 14 and 15, and Brasmid pME. Fig. 9 shows the construction steps of 18 S / mF as and EXT, and the cDNA sequence encoding mF as ■ EXT and the corresponding amino acid sequence are shown in SEQ ID NOs: 16 and 17, respectively.

<9-3> Acquisition of cDNA comprising mFas * EXT and cDNA encoding AIC2A extracellular region (AIC2A * EXT)

 The plasmid p CEV4ZA constructed in <8-3-1-F> was digested with Drain to extract cDNA encoding AIC2AEXT, and the fragment was digested using a DNA-end blunting kit. The cut end was blunted and treated with BAP to remove the phosphate group at the 5 'end of the cut surface.

 On the other hand, the linker DNA sequence of the plasmid pME18S / mFas.EXT constructed in 9-2> was digested with DraIII, and the DNA ligation kit was used to cut the Dralll-cut AIC2AEXT. The cDNA fragment was ligated to the DrAIII cleavage linker DNA to construct the plasmid pME18S / mFas-EXT-AIC2A.EXT.

 Figure 10 shows the construction process of ME 18 S / mF as and EXT-A IC 2 A and EXT.The cDNA sequence and the corresponding amino acid sequence encoding mF as-EXT-A IC 2 A and EXT are respectively shown. These are shown in SEQ ID NOs: 18 and 19.

 <9-4> Plasmid of E. coli cell line JM109 pME18S / mFas Transformation with EXT-AIC2AEXT

 Transform the plasmid pME18S / mFas-EXT-AIC2A.EXT constructed in Step 9 into the E.co1 i cell line JM109 and analyze the colonies for transformation. Cells were selected. The transformed E. coli cell strain JM109 was deposited internationally with the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Accession number: FERM BP-44437, date of acceptance: 199) 3 years 1 October 6).

Expression, purification and analysis of chimeric soluble mouse Fas antigen (mFas-AIC2A)

Transformed cells JM109 to plasmid pME18S / FasEXT-A IC 2 A · EXT was taken out by a conventional method, and C 0 S 7 was transformed with the plasmid by the electroporation method (see Nucl. Acids Res., Vol. 16, pp. 6127-6145, 1988, Dower et al.). did.

 After the transformed COS 7 is cultured in D-MEM medium containing 10% FCS for 45 hours, the serum-free culture obtained by culturing in serum-free D-MEM medium for 12 hours is centrifuged and centrifuged. Collected.

 The obtained centrifuged supernatant was analyzed by SDS-polyacrylamide gel electrophoresis, and as a result, it was confirmed that the obtained soluble protein was obtained as a soluble mFas-AIC2A protein. The centrifuged supernatant was subjected to column chromatography using Q Sepharose to purify a soluble protein comprising an extracellular region of mFas and an extracellular region of AIC2A. The results are shown in FIG.

Example 10 Production of rat anti-mouse Fa s monoclonal antibody (rat anti-mFa s MAb)

<10-1> Preparation of antibody-producing cells

Chimeric soluble mouse Fas antigen (soluble mFa s—AIC 2 A) (100 g / PBS) and killed bacteria (2 X 10 ^s ) of Bordetella pertussis purified by <9-5> Was administered intraperitoneally to Lewis rats, and on the 14th day, soluble mFas-AI C2A (10 X £ g / PBS) was further intraperitoneally administered. Three days after the administration, the rats were laparotomized, the spleens were excised, triturated on a stainless steel mesh in a serum-free R PMI 1640 culture solution, and the spleen cell solution was centrifuged (7 minutes at 1500 rpm). The centrifuged residue was collected and suspended in a bloodless RPMI 1640 culture solution. Furthermore, the cells were washed twice with a serum-free RPMI 1640 culture solution to obtain antibody-producing rat spleen cells.

 <10-2> Preparation of mouse myeloma pulmonary vesicles

The mouse myeloma Tsumugi胞NS- 1 (ATCC TIB18), 37 '(:, 5¾C0 2 below, were cultured in 1 0% FCS and 5 OUZml kanamycin ASF- 1 04 medium (Ajinomoto (Ltd.)).

<10-3> Preparation of anti-mouse Fas monoclonal antibody-producing hybridoma Mouse myeloma cells NS-1 cells washed with serum-free RPMI 1640 medium

(3 x 10 ⁷ ) and rat spleen cells (3 x 10 ⁸ ) prepared in <10-1> were mixed and centrifuged at 10,000 rpm for 10 min. Then, the centrifugation residue was collected, and cell fusion was performed according to the method of Herzenberg, LA (La.) Et al. (Selected Methods in Cellular Immunology. P.351, 1980) to obtain a plurality of colonies of hybridomas.

 Culture of each hybridoma colony against Ba1b / c mouse thymocytes expressing the Fas antigen in order to screen the hybridoma producing the anti-mouse Fas monoclonal antibody. The sex was measured by flow cytometry, and four hybridoma clones producing anti-mouse Fas monoclonal antibodies, RMF2, RMF6, RMF9 and RMF13 were obtained. Clone RMF 6 has been deposited internationally with the Institute of Biotechnology, Ministry of International Trade and Industry of Japan (Accession number: FERM BP-4394, Accession date: August 25, 1999).

<10-4> Obtaining rat anti-mouse F a s monoclonal antibody

 Each of the hybridomas was cultured in a serum-free ASF104 culture medium, and the culture supernatant was centrifuged (8000 rpm for 20 minutes). The centrifuged supernatant was recovered, and each monoclonal antibody was purified by column chromatography using Protein G Sepharose (Pharmacia).

 In addition, rat monoclonal antibody subclass identification kit (Zymed)

As a result of analyzing each monoclonal antibody subtype using (manufactured)), RMF2 was IgG1, RMF6 was IgG2a, RMF9 was IgG2a, and RMF13 was

It was confirmed to be IgM.

Example 11 Production of Hamster Anti-Mouse F aS Monoclonal Antibody (Hamster Anti-mF a sMAb)

 <11-1> Preparation of antibody-viable cells

<9 one 5> purified acquired chimeric soluble murine F the as antigens (soluble mF as- AIC 2 A) (1 00〃 g / PBS) and dead bacteria (2 X 1 0 ⁹ cells) of B. pertussis (Bordetella pertussis) Is administered intraperitoneally to Armenian hamsters, On the 14th day, soluble mFas-AIC 2A (10 / xg / PBS) was further intraperitoneally administered. Three days after administration, the hamster was laparotomized, the spleen was excised, ground in a serum-free RPMI 1640 culture medium on a stainless steel mesh, and the spleen cell suspension was centrifuged.

(At 1500 rpm for 7 minutes). The centrifugation residue was collected and suspended in serum-free RPMI 1640 medium. Furthermore, the cells were washed twice with a serum-free RPMI 1640 culture solution to obtain antibody-producing hamus spleen cells.

<11-2> Preparation of mouse myeloma cells

The mouse myeloma cells NS- 1 (ATCC TIB18), at 37, under _{5% C0 2, 1 0%} FCS and 50 U / m 1 kanamycin-containing ASF- 1 04 medium (Ajinomoto

(Manufactured)).

Ku 1 1 - 3> anti-mouse F the as monoclonal antibody producing High Priestess dormer mouse myeloma cells NS prepared were washed with serum-free RPM 1 1 640 broth - 1 cells (3 X 1 0 ⁷ pieces) and <1 1 one 1> in prepared hamster splenocytes (3 X 1 0 ⁸ cells) centrifuged serum RPMI 1 64 0 a mixed solution of (1 0 min 1 000 r pm) and collected by centrifugation residue, Hell Tseng Berg (Herzenberg Cell fusion was carried out in accordance with the method of S. Shi, et al. (Selected Methods in Cellular Immunology. P.351, 1980) to obtain a plurality of hybridoma colonies.

 In order to screen for hybridomas producing anti-mouse Fas monoclonal antibodies, the reactivity of the culture supernatant of each hybridoma colony against Ba1c mouse thymocytes expressing the Fas antigen was determined. As measured by flow cytometry, four hybridoma clones producing anti-mouse Fas monoclonal antibodies, RK-8, SK-8.C6-1, and P4-4, were obtained. The clones RK-8 and SK-8 have been internationally deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, respectively (accession number: FERM BP-4756, accession date: July 26, 1994, And accession number: FERM BP-4667, accession date: stated May 16, 1993).

 <11-4> Acquisition of hamster anti-mouse Fas monoclonal antibody

Culture each of the hybridomas in serum-free ASF104 culture medium, Was centrifuged (8000 rpm for 20 minutes). The centrifuged supernatant was collected, and each monoclonal antibody was purified by column chromatography using Protein A Sepharose (Pharmacia).

Example 12 Test for Confirming Reactivity to Mouse Fs Antigen

 The reactivity of the rat anti-mouse Fas monoclonal antibody and hamster anti-mouse Fas monoclonal antibody obtained in Example 10 and Example 11 respectively with mouse Fa antigen was confirmed by flow cytometry as follows.

 The reactivity was determined by introducing the mouse lymphoma cell L5178Y (ATCC CRL1722) which does not express the Fas antigen and the full-length mouse Fas cDNA obtained by 9-1-1-C> by gene engineering. It was confirmed by comparing the reactivity to the L5178Y cells overexpressing the mouse Fas antigen. The mouse Fa s antigen overexpressing L5178 Y cells were prepared as follows.

 The plasmid pMF1 containing the full-length mouse Fas cDNA obtained in <9-11C> was digested with EcoR1, and the cut ends were digested using a DNA end blunting kit (Takara Shuzo Co., Ltd.). Was smoothed to obtain a DNA fragment containing cDNA encoding full-length mouse Fas cDNA. On the other hand, the expression vector pME183 described above in <8-1-1C> was digested with 110 I, and the cut ends were blunted using a DNA end blunting kit (Takara Shuzo Co., Ltd.). The l8S fragment was obtained. The pMF1 fragment having the full-length mouse Fas cDNA and the pME18S fragment were ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.) to obtain a plasmid pME18S / mFas having the full-length mouse Fas cDNA. It was constructed.

This pME18S / mFas was digested with PvuI by electroporation (290 V) using Gene Pulser (manufactured by Bio-Rad) to obtain pME18S / mF. A mouse lymphoma cell L5178Y (ATCCCRL1722) was co-transformed with the as fragment and a plasmid p MAMneo (Clontech) fragment obtained by digestion with EcoRI. The transformed cells were cultured in a medium containing the antibiotic G418 to select for G418-resistant clones and by limiting dilution to obtain mouse Fas antigen-overexpressing L5178Y cells. Mouse lymphoma cells L5178 Y not expressing the Fas antigen or the suspension of the mouse Fs antigen overexpressing L5178 YB vesicle were added to the suspension obtained in Examples 10 and 11, respectively. PBS containing the anti-mouse Fas monoclonal antibody (20 PL g / m 1) was added, and the mixture was allowed to react on water for 1 hour. Next, PBS containing FITC-labeled anti-rat IgG, FITC-labeled anti-hamster IgG or FITC-labeled anti-rat IgM (each 20 gZml) was added and reacted for 1 hour. After washing each cell with PBS (with 4), the reactivity was analyzed by cytofluorimetry.

 As a control, anti-rat IgG, anti-hamster IgG and anti-rat IgM (Pharraingen) were similarly measured.

 The results are shown in FIGS. 12 and 13.

It was confirmed that each of the obtained monoclonal antibodies was a monoclonal antibody having specific reactivity to mouse Fas antigen.

(Sequence list)

Sequence number (SEQ ID NO): 1

Sequence length (SEQUENCE LENGTH): 12

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

 Topology (TOPOLOGY): linear

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 CTTTAGAGCA CA 12 SEQ ID NO: 2

Sequence length (SEQUENCE LENGTH): 2534

Sequence type (SEQUENCE TYPE): nucleic acid

STRANDNESS: Double strand

 Topology (TOPOLOGY): Straight

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 GACGCTTCTG GGGAGTGAGG GAAGCGGTTT ACGAGTGACT TGGCTGGAGC CTCAGGGGCG 60 GGCACTGGCA CGGAACACAC CCTGAGGCCA GCCCTGGCTG CCCAGGCGGA GCTGCCTCTT 120 CTCCCGCGGG TTGGTGGACC CGCTCAGTAC GGAGTTGGGG AAGCTCTTTC ACTTCGGAGG 180 ATTGCTCAAC AACC ATG CTG GGC ATC TGG ACC CTC CTA CCT CTG GTT CTT 230

 Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu

 -15 -10 -5

 ACG TCT GTT GCT AGA TTA TCG TCC AAA ACT GTT AAT GCC CAA GTG ACT 278 Thr Ser Val Ala Arg Leu Ser Ser Lys Ser Val Asn Ala. Gin Val Thr

 1 5 10

 GAC ATC AAC TCC AAG GGA TTG GAA TTG AGG AAG ACT GTT ACT ACA GTT 326 Asp lie Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val

15 20 25 GAG ACT CAG AAC TTG GAA GGC CTG CAT CAT GAT GGC CAA TTC TGC CAT 374 Glu Thr Gin Asn Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His

 30 35 40

 AAG CCC TGT CCT CCA GGT GAA AGG AAA GCT AGG GAC TGC ACA GTC AAT 422 Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn

 45 50 55 60

 GGG GAT GAA CCA GAC TGC GTG CCC TGC CAA GAA GGG AAG GAG TAC ACA 470 Gly Asp Glu Pro Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr

 65 70 75

 GAC AAA GCC CAT TTT TCT TCC AAA TGC AGA AGA TGT AGA TTG TGT GAT 518 Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp

 80 85 90

 GAA GGA CAT GGC TTA GAA GTG GAA ATA AAC TGC ACC CGG ACC CAG AAT 566 Glu Gly His Gly Leu Glu Val Glu lie Asn Cys Thr Arg Thr Gin Asn

 95 100 105

 ACC AAG TGC AGA TGT AAA CCA AAC TTT TTT TGT AAC TCT ACT GTA TGT 614 Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys

 110 115 120

 GAA CAC TGT GAC CCT TGC ACC AAA TGT GAA CAT GGA ATC ATC AAG GAA 662 Glu His Cys Asp Pro Cys Thr Lys Cys Glu His Glylie He Lys Glu

125 130 135 140

 TGC ACA CTC ACC AGC AAC ACC AAG TGC AAA GAG GAA GGA TCC AGA TCT 710 Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser

 145 150 155

 AAC TTG GGG TGG CTT TGT CTT CTT CTT TTG CCA ATT CCA CTA ATT GTT 758 Asn Leu Gly Trp Leu Cys Leu Leu Leu Leu Pro lie Pro Leu lie Val

 160 165 170

 TGG GTG AAG AGA AAG GAA GTA CAG AAA ACA TGC AGA AAG CAC AGA AAG 806

5 o Trp Val Lys Arg Lys Glu Val Gin Lys Thr Cys Arg Lys His Arg Lys

 175 180 185

 GAA AAC CAA GGT TCT CAT GAA TCT CCA ACC TTA AAT CCT GAA ACA GTG 854 Glu Asn Gin Gly Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val

 190 195 200

 GCA ATA AAT TTA TCT CAT GTT GAC TTG AGT AAA TAT ATC ACC ACT ATT 902 Ala lie Asn Leu Ser Asp Val Asp Leu Ser Lys Tyr lie Thr Thr lie

205 210 215 220

 GCT GGA GTC ATG ACA CTA AGT CAA GTT AAA GGC TTT GTT CGA AAG AAT 950 Ala Gly Val Met Thr Leu Ser Gin Val Lys Gly Phe Val Arg Lys Asn

 225 230 235

 GGT GTC AAT GAA GCC AAA ATA GAT GAG ATC AAG AAT GAC AAT GTC CAA 998 Gly Val Asn Glu Ala Lys He Asp Glu lie Lys Asn Asp Asn Val Gin

 240 245 250

 GAC ACA GCA GAA CAG AAA GTT CAA CTG CTT CGT AAT TGG CAT CAA CTT 1046 Asp Thr Ala Glu Gin Lys Val Gin Leu Leu Arg Asn Trp His Gin Leu

 255 260 265

 CAT GGA AAG AAA GAA GCG TAT GAC ACA TTG ATT AAA GAT CTC AAA AAA 1094 His Gly Lys Lys Glu Ala Tyr Asp Thr Leu lie Lys Asp Leu Lys Lys

 270 275 280

 GCC AAT CTT TGT ACT CTT GCA GAG AAA ATT CAG ACT ATC ATC CTC AAG 1142 Ala Asn Leu Cys Thr Leu Ala Glu Lys He Gin Thr lie lie Leu Lys

285 290 295 300

 GAC ATT ACT AGT GAC TCA GAA AAT TCA AAC TTC AGA AAT GAA ATC CAA 1190 Asp lie Thr Ser Asp Ser Glu Asn Ser Asn Phe Arg Asn Glulie Gin

 305 310 315

AGC TTG GTC TAGAGTGAAA AACAACAAAT TCAGTTCTGA GTATATGCAA TTAGTGTTTG 1249 Ser Leu Val AAAAGATTCT TAATAGCTGG CTGTAAATAC TGCTTGGTTT TTTACTGGGT ACATTTTATC 1309

ATTTATTAGC GCTGAAGAGC CAACATATTT GTAGATTTTT AATATCTCAT GATTCTGCCT 1369

CCAAGGATGT TTAAAATCTA GTTGGGAAAA CAAACTTCAT CAAGAGTAAA TGCAGTGGCA 1429

TGCTAAGTAC CCAAATAGGA GTGTATGCAG AGGATGAAAG ATTAAGATTA TGCTCTGGCA 1489

TCTAACATAT GATTCTGTAG TATGAATGTA ATCAGTGTAT GTTAGTACAA ATGTCTATCC 1549

ACAGGCTAAC CCCACTCTAT GAATCAATAG AAGAAGCTAT GACCTTTTGC TGAAATATCA 1609

GTTACTGAAC AGGCAGGCCA CTTTGCCTCT AAATTACCTC TGATAATTCT AGAGATTTTA 1669

CCATATTTCT AAACTTTGTT TATAACTCTG AGAAGATCAT ATTTATGTAA AGTATATGTA 1729

TTTGAGTGCA GAATTTAAAT AAGGCTCTAC CTCAAAGACC TTTGCACAGT TTATTGGTGT 1789

CATATTATAC AATATTTCAA TTGTGAATTC ACATAGAAAA CATTAAATTA TAATGTTTGA 1849

CTATTATATA TGTGTATGCA TTTTACTGGC TCAAAACTAC CTACTTCTTT CTCAGGCATC 1909

AAAAGCATTT TGAGCAGGAG AGTATTACTA GAGCTTTGCC ACCTCTCCAT TTTTGCCTTG 1969

GTGCTCATCT TAATGGCCTA ATGCACCCCC AAACATGGAA ATATCACCAA AAAATACTTA 2029

ATAGTCCACC AAAAGGCAAG ACTGCCCTTA GAAATTCTAG CCTGGTTTGG AGATACTAAC 2089

TGCTCTCAGA GAAAGTAGCT TTGTGACATG TCATGAACCC ATGTTTGCAA TCAAAGATGA 2149

TAAAATAGAT TCTTATTTTT CCCCCACCCC CGAAAATGTT CAATAATGTC CCATGTAAAA 2209

CCTGCTACAA ATGGCAGCTT ATACATAGCA ATGGTAAAAT CATCATCTGG ATTTAGGAAT 2269

TGCTCTTGTC ATACCCTCAA GTTTCTAAGA TTTAAGATTC TCCTTACTAC TATCCTACGT 2329

TTAAATATCT TTGAAAGTTT GTATTAAATG TGAATTTTAA GAAATAATAT TTATATTTCT 2389

GTAAATGTAA ACTGTGAAGA TAGTTATAAA CTGAAGCAGA TACCTGGAAC CACCTAAAGA 2449

ACTTCCATTT ATGGAGGATT TTTTTGCCCC TTGTGTTTGG AATTATAAAA TATAGGTAAA 2509

ACTACGTAAT TAAATAATGT TTTTG 2534 SEQ ID NO: 3

Sequence length (SEQUENCE LENGTH): 335

Sequence type (SEQUENCE TYPE): amino acid

Sequence type (MOLECULE TYPE): Protein

Sequence (SEQUENCE DESCRIPTION):

Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala -15 -10 -5

 Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp lie Asn Ser

1 5 10 15

Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn

 20 25 30

 Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro

 35 40 45

 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro

50 55 60

 Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 65 70 75 80

Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly

 85 90 95

Leu Glu Val Glu lie Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg

 100 105 110

 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp

 115 120 125

 Pro Cys Thr Lys Cys Glu His Gly lie lie Lys Glu Cys Thr Leu Thr

130 135 140

 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 145 150 155 160

Leu Cys Leu Leu Leu Leu Pro lie Pro Leu He Val Trp Val Lys Arg

 165 170 175

Lys Glu Val Gin Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gin Gly

 180 185 190

 Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala 〖ie Asn Leu

 195 200 205

Ser Asp Val Asp Leu Ser Lys Tyr l ie Thr Thr lie Ala Gly Val Met 210 215 220

 Thr Leu Ser Gin Val Lys Gly Phc Val Arg Lys Asn Gly Val Asn Glu

225 280 235 240

 Ala Lys li e Asp filti M P. i.ys Asn Asp Asn Val Gin Asp Thr Ala Glu

 245 250 255

 Gin Lys Val Gin Leu Leu Arg Asn TI P His Glu Leu His Gly Lys Lys

 260 265 270

 Glu Ala Tyr Asp Thr Leu lie Lys Asp Leu Lys Lys Ala Asn Leu Cys

 275 280 285

 Thr Leu Ala Glu Lys lie Gin Thr He lie Leu Lys Asp lie Thr Ser

 290 295 SOO

 Asp Ser Glu Asn Ser Asn Phe Arg Asn Glulie Gin Ser Leu Val

305 810 315

 Sequence number (SEQ ID NO): i

Good sequence (SEQUENCE LENGTH): 519

Sequence 5? (SEQUENCE TYPE): Nucleic acid

Number of chains (STKANDNIiSS): Niki

 Topology: TOPOLOGY

OIOLECULE TYPE): cDNA

'Distribution evening'! SKQUIiNCfa 'UliSCHlPnON):

 ATG CTG GGC ATC TGG ACC CTC CTA CCT CTG GTT CTT ACG TCT GTT GCT 48 Met Leu Gly He Trp Thr I.eu l.eu Pro Leu Val Leu Thr Ser Val Ala

 -15 -10 -5

 AGA TTA TCG TCC AAA AGT GTT AAT GCC CAA GTG ACT GAC ATC AAC TCC 96 Arg Leu Ser Ser lys Ser Val Asn Ala Gin Val Thr Asp lie Asn Ser

 1 5 10 15

AAG GGA TTG GAA TTG AGG AAG ACT GTT ACT ACA GTT GAG ACT CAG AAC 144 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Giu Thr Gin Asn ε s 翘 丄 ·· OdAl 33N3n¾aS) fil <2) iii2i δλΐ ： (Η10Ν3Ί 30N3nb3S)? ¥ C2) M2i

 9 (ON αΐ5HS) ¼ # 2S ssi ogi g ^ t usv J8S 3JV i3S Aio nig nig sxq SAQ sxq jqi asy J9S 19 OVV 1D1 VOV 031 VOD VVO OVO VVV DDI 3VV 33V OVV OOV

 OH 981 OSI

J¾ naq J¾ SAQ nig sAq an an Aig S IH SAQ s jqi sAo OJJ 8 ^ 33V 013 VOV 301 VV3 OVV 31V 01V V99 IVO VV9 101 VVV OOV 001 130

 9ZI 021 911 dsy sxo S IH nio s ^ o Ι¾Λ J¾ «sy SXQ aqj av ^ usy OJJ sAq SAQ

ZS OVO 101 OVO VVO 131 VIO IOV 101 OVV 101 III 111 OVV VOO VVV 131

 Oil 901 001

 8 V SAQ sAq J¾ usv UI9 3jy J¾ SAQ USV 311 nion niD naq VOV 301 OVV 33V IVV OVO 33V 393 03V 301 OVV VIV VVO 010 VVO Vll S6 06 98

 Aio S IH A I D "10 dsy SAQ naq 3jy s 3jy 8jy SAQ sXq Jas J9S shout d9S8 000 IVO VDO VVO IVO 101 Oil VOV 101 VOV VDV 001 VVV 001 101 111

 08 Si 02. 99

S IH ¾ IV s 1 dsy “ェ” 10 s Aionio UIQ SAQ o ^ d Ι ¾Λ sAQ dsy 882 1V3 009 VVV OVO VOV OVl OVO OVV 093 VVO VV3 001 333 910 001 001 OVO

 09 SS OS

OJd nig dsv ^ io usy ^JL U δ 3 dsy 3iy ¾IV sAq 8JV io oid VOO VVO IVO 300 IVV 310 VOV 091 OVO 30V 130 VVV OOV VVO 100 V30 gt-98 oid sA3 oid sXq S IH SAQ aqj uig S IH nsi Aio nig na

361 130 101 000 OVV IVO 001 Oil VV3 090 IVO IVO IVO 013 300 VVO Oil

 08 92 02 0/96 O

6½00 / S6dT / 13d Sequence type (MOLECULE TYPE): Protein

Sequence (SEQUENCE DESCRIPTION):

 Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala

-15 -10 -5

 Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp He Asn Ser

1 5 10 15

Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn

 20 25 30

 Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro

 35 40 45

 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro

50 55 60

 Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 65 70 75 80

Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly

 85 90 95

Leu Glu Val Glu He Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg

 100 105 110

 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp

 115 120 125

 Pro Cys Thr Lys Cys Glu His Gly lie lys Lys Glu Cys Thr Leu Thr

130 135 140

 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn

145 150 155

SEQ ID NO: 6

Sequence length (SEQUENCE LENGTH): 3001

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand Topology (TOPOLOGY): linear

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 AGGCTGCCTG TCGCCCAAGC ACAGAGCCAC AAAGGATGCA GTCTAGGAGG GAAGAATCAC 60 AAGCCCTGTA AGATGAGTGG AGCCAAACCC CCAGCCAAGC ACCAATACAG AACCCCGGGT 120 CAATGAGGAC ACCCCCCTGC CCATAGCTTC CTGTGCAGCC ACCAAAAGTG CCAAA ATG 178

 Met

 GAC CAG CAA ATG GCA CTC ACA TGG GGG CTG TGC TAC ATG GCA CTG GTG 226 Asp Gin Gin Met Ala Leu Thr Trp Gly Leu Cys Tyr Met Ala Leu Val

 -20 -15 -10

 GCT CTC TGC TGG GGA CAC GAG GTG ACA GAG GAA GAA GAA ACG GTC CCT 274 Ala Leu Cys Trp Gly His Glu Val Thr Glu Glu Glu Glu Thr Val Pro

 -5 1 5 10

 CTG AAG ACT CTG GAG TGC TAC AAT GAC TAC ACC AAC CGT ATC ATC TGC 322 Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr Asn Arglie He Cys

 15 20 25

 AGC TGG GCA GAC ACA GAG GAT GCC CAG GGG CTA ATC AAC ATG ACC CTC 370 Ser Trp Ala Asp Thr Glu Asp Ala Gin Gly Leu He Asn Met Thr Leu

 30 35 40

 CTC TAT CAC CAG CTA GAC AAG ATT CAA TCA GTG TCC TGT GAG CTC ACT 418 Leu Tyr His Gin Leu Asp Lys lie Gin Ser Val Ser Cys Glu Leu Ser

 45 50 55

 GAG AAA CTC ATG TGG TCA GAG TGC CCG TCA TCC CAC CGC TGT GTG CCC 466 Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val Pro

 60 65 70 75

 AGA AGA TGT GTC ATC CCC TAC ACA CGA TTT TCT AAT GGA GAT AAC GAC 514 Arg Arg Cys Val lie Pro Tyr Thr Arg Phe Ser Asn Gly Asp Asn Asp

80 85 90 TAC TAC TCC TTC CAG CCA GAT CGT GAC CTG GGC ATC CAG CTC ATG GTC 562 Tyr Tyr Ser Phe Gin Pro Asp Arg Asp Leu Gly He Gin Leu Met Val

 95 100 105

 CCA CTG GCC CAG CAT GTG CAG CCA CCC CCT CCC AAG GAC ATC CAC ATC 610 Pro Leu Ala Gin His Val Gin Pro Pro Pro Pro Lys Asp lie His lie

 110 115 120

 AGC CCC TCT GGG GAT CAT TTC CTG CTG GAA TGG AGT GTA TCT CTT GGG 658 Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu Gly

 125 130 135

GAT TCC CAG GTC TCC TGG CTT TCA TCA AAG GAC ATA GAG TTT GAG GTG 706 Asp Ser Gin Val Ser Trp Leu Ser Ser Lys Asplie Glu Phe Glu Val

140 145 150 155

 GCT TAT AAG CGG CTT CAG GAC TCC TGG GAG GAT GCC TCC AGT CTC CAC 754 Ala Tyr Lys Arg Leu Gin Asp Ser Trp Glu Asp Ala Ser Ser Leu His

 160 165 170

 ACT AGC AAC TTT CAG GTG AAT TTA GAG CCA AAG CTA TTC CTA CCC AAC 802 Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys Leu Phe leu Pro Asn

 175 180 185

 AGC ATC TAT GCT GCC CGT GTG CGC ACT CGG CTG TCC GCG GGT TCA AGC 850 Ser lie Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser Ser

 190 195 200

 TTG TCT GGG AGA CCC AGC AGA TGG AGC CCA GAG GTT CAC TGG GAC TCC 898 Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu Val His Trp Asp Ser

 205 210 215

 CAG CCA GGG GAC AAG GCC CAG CCA CAG AAC CTT CAA TGC TTC TTT GAT 946 Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu Gin Cys Phe Phe Asp

220 225 230 235

GGG ATC CAG TCT CTC CAC TGC TCC TGG GAG GTG TGG ACC CAG ACG ACT 994 Gly He Gin Ser Leu His Cys Ser Trp Glu Val Trp Thr Gin Thr Thr

 240 245 250

 GGC TCT GTT TCC TTT GGG CTC TTC TAT CGC CCC AGC CCT GCA GCT CCG 1042 Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro Ser Pro Ala Ala Pro

 255 260 265

 GAG GAG AAA TGC TCT CCG GTG GTG AAG GAG CCG GAG GCC AGC GTC TAC 1090 Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val Tyr

 270 275 280

 ACC CGC TAC CGC TGC ACT CTA CCT GTG CCT GAG CCC ACT GCA CAC AGC 1138 Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu Pro Ser Ala His Ser

 285 290 295

 CAG TAC ACA GTC TCT GTT AAG CAC CTG GAA CAA GGG AAG TTC ATC ATG 1186 Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin Gly Lys Phe lie Met

300 305 310 315

 AGC TAT TAC CAC ATC CAG ATG GAA CCT CCA ATC CTC AAC CAG ACC AAG 1234 Ser Tyr Tyr His lie Gin Met Glu Pro Pro lie Leu Asn Gin Thr Lys

 320 325 330

 AAC AGA GAC AGC TAC AGC CTG CAT TGG GAA ACT CAG AAA ATA CCC AAA 1282 Asn Arg ASP Ser Tyr Ser Leu His Trp Glu Thr Gin Lys 〖le Pro Lys

 335 340 345

 TAC ATT GAT CAC ACT TTC CAG GTC CAG TAC AAG AAG AAG TCA GAG AGC 1330 Tyr l ie Asp His Thr Phe Gin Val Gin Tyr Lys Lys Lys Ser Glu Ser

 S50 355 360

 TGC AAG GAC AGC AAG ACA GAA AAC CTA GGT CGA GTC AAT AGC ATG GAC 1378 Trp Lys Asp Ser Lys Thr Glu Asn Leu Gly Arg Val Asn Ser Met Asp

 365 370 375

CTG CCC CAG CTG GAG CCG GAC ACC TCA TAC TGC GCC AGG GTG AGG GTC 1426 Leu Pro Gin Leu Glu Pro Asp Thr Ser Tyr Cys Ala Arg Val Arg Val 380 385 390 395

 AAG CCC ATC TCT GAC TAC CAC GGG ATC TGG AGC GAG TGG AGC AAT GAG 1474 Lys Pro lie Ser Asp Tyr ASP Gly He Trp Ser Glu Trp Ser Asn Glu

 400 405 410

 TAC ACT TGG ACG ACT GAC TGG GTG ATG CCC ACG CTG TGG ATA GTC CTC 1522 Tyr Thr Trp Thr Thr Asp Trp Val Met Pro Thr Leu Trp lie Val Leu

 415 420 425

 ATC CTG GTC TTT CTC ATC TTC ACC TTC CTC CTG GCT CTC CAC TTT GGC 1570 He Leu Val Phe Leu lie Phe Thr Leu Leu Leu Ala Leu His Phe Gly

 430 435 440

 CGT GTT TAT GGG TAC AGG ACA TAC AGG AAG TGG AAG GAA AAC ATC CCC 1618 Arg Val Tyr Gly Tyr Arg Thr Tyr Arg Lys Trp Lys Glu Lys lie Pro

 445 450 455

 AAC CCC AGC AAG AGC CTC CTG TTC CAG GAT GGA GGT AAA GGT CTC TGG 1666 Asn Pro Ser Lys Ser Leu Leu Phe Gin Asp Gly Gly Lys Gly Leu Trp

460 465 470 475

 CCT CCT GGC ACC ATG GCA GCC TTC GCG ACT AAG AAC CCC GCT CTC CAG 1714 Pro Pro Gly Ser Met Ala Ala Phe Ala Thr Lys Asn Pro Ala Leu Gin

 480 485 490

 GGG CCA CAG AGC AGG CTT CTT CCT GAG CAA CAG GGG GTG TCA TAT GAA 1762 Gly Pro Gin Ser Arg Leu Leu Ala Glu u Gin Gin Gly Val Ser Tyr Glu

 495 500 505

 CAT TTG GAA GAC AAC AAC GTG TCA CCT CTC ACT ATA GAG GAC CCT AAT 1810 His Leu Glu Asp Asn Asn Val Ser Pro Leu Thr lie Glu Asp Pro Asn

 510 515 520

 ATA ATT CGA GAT CCA CCA TCC AGG CCT GAT ACA ACC CCA GCT GCC TCA 1858 lie lie Arg Asp Pro Pro Ser Arg Pro Asp Thr Thr Pro Ala Ala Ser

525 530 535 TCT GAA TCC ACA GAG CAA CTT CCC AAT GTT CAA GTA GAG GGA CCA ATT 1906

Ser Glu Ser Thr Glu Gin Leu Pro Asn Val Gin Val Glu Gly Pro He

 540 545 550 555

 CCT TCT AGC CGA CCC AGG AAG CAA TTA CCC AGC TTT GAC TTC AAT GGG 1954

Pro Ser Ser Arg Pro Arg Lys Gin Leu Pro Ser Phe Asp Phe Asn Gly

 560 565 570

 CCC TAC CTG GGG CCT CCC CAA TCC CAC TCC CTG CCT GAT CTC CCA GGC 2002 Pro Tyr Leu Gly Pro Pro Gin Ser His Ser Leu Pro Asp Leu Pro Gly

 575 580 585

 CAG CTG GGT TCT CCC CAG CTG GGT GGG AGC CTG AAG CCA GCA CTG CCA 2050 Gin Leu Gly Ser Pro Gin Val Gly Gly Ser Leu Lys Pro Ala Leu Pro

 590 595 600

 GGC TCC TTG GAG TAC ATG TGT CTG CCC CCT GGA GGT CAA GTG CAA CTG 2098 Gly Ser Leu Glu Tyr Met Cys Leu Pro Pro Gly Gly Gin Val Gin Leu

 605 610 615

 GTC CCA TTG TCC CAG GTG ATG GGA CAG GGC CAG GCT ATG GAT GTG CAG 2146 Val Pro Leu Ser Gin Val Met Gly Gin Gly Gin Ala Met Asp Val Gin

620 625 630 635

 TGT GGG TCC AGC CTG GAG ACC ACA GGG AGC CCT TCC GTG GAG CCA AAG 2194 Cys Gly Ser Ser Leu Glu Thr Thr Gly Ser Pro Ser Val Glu Pro Lys

 640 645 650

 GAG AAC CCT CCA GTT GAG CTG AGC GTG GAG AAA CAG GAG GCA AGG GAC 2242 Glu Asn Pro Pro Val Glu Leu Ser Val Glu Lys Gin Glu Ala Arg Asp

 655 660 665

 AAC CCA ATG ACT CTT CCC ATA AGC TCT GGG GGC CCT GAG GGC AGT ATG 2290 Asn Pro Met Thr Leu Pro lie Ser Ser Gly Gly Pro Glu Gly Ser Met

 670 675 680

ATG GCC TCT GAT TAT GTC ACT CCT GGA GAT CCG GTG CTC ACT CTG CCC 2338 Met Ala Ser Asp Tyr Val Thr Pro Gly Asp Pro Val Leu Thr Leu Pro

 685 690 695

 ACA GGG CCC CTG TCT ACC TCT CTG GGC CCC TCT CTA GGG TTG CCC TCA 2386 Thr Gly Pro Leu Ser Thr Ser Leu Gly Pro Ser Leu Gly Leu Pro Ser

700 705 710 715

 GCC CAA AGC CCC AGT CTC TGT CTT AAG CTG CCC AGG GTC CCC TCT GGA 2434 Ala Gin Ser Pro Ser Leu Cys Leu Lys Leu Pro Arg Val Pro Ser Gly

 720 725 730

 AGC CCA GCT CTA GGG CCA CCA GGG TTT GAG GAC TAT GTG GAG CTG CCT 2482 Ser Pro Ala Leu Gly Pro Pro Gly Phe Glu Asp Tyr Val Glu Leu Pro

 735 740 745

 CCA AGT GTG AGC CAG GCA GCC ACG TCC CCT CCA GGC CAT CCT GCT CCT 2530 Pro Ser Val Ser Gin Ala Ala Thr Ser Pro Pro Gly His Pro Ala Pro

 750 755 760

 CCT GTG GCA AGC AGC CCC ACA GTG ATC CCA GGA GAG CCC AGG GAG GAA 2578 Pro Val Ala Ser Ser Pro Thr Val lie Pro Gly Glu Pro Arg Glu Glu

 765 770 775

 GTG GGC CCA GCA TCC CCA CAT CCC GAA GGC CTC CTT GTT CTT CGG CAG 2626 Val Gly Pro Ala Ser Pro His Pro Glu Gly Leu Leu Val Leu Arg Gin

780 785 790 795

 GTT GGG GAC TAC TGC TTC CTC CCT GGC CTG GGA CCT GGC TCC CTC TCA 2674 Val Gly Asp Tyr Cys Phe Leu Pro Gly Leu Gly Pro Gly Ser Leu Ser

 800 805 810

 CCA CAC AGT AAG CCA CCC TCT CCA AGT CTG TGT TCT GAG ACT GAG GAC 2722 Pro His Ser Lys Pro Pro Ser Pro Ser Leu Cys Ser Glu Thr Glu Asp

 815 820 825

CTA GAC CAG GAC TTG TCT GTG AAA AAG TTT CCC TAT CAA CCC TTG CCC 2770 Leu Asp Gin Asp Leu Ser Val Lys Lys Phe Pro Tyr Gin Pro Leu Pro 830 835 840

 CAG GCC CCA GCC ATT CAG TTT TTC AAG TCC CTA AAG TAT TAGGACTACC 2819 Gin Ala Pro Ala lie Gin Phe Phe Lys Ser Leu Lys Tyr

 845 850 855

 TGTCCCTGCC CCCTTGGGAC AATAGCCAGT CTGGGAAGGT GTGCTGAGTC TGTCCCCTCC 2879 CAATCTCACC AGCAGCCTGG CACCGCAGCC TGTGGTCCTC AGCCTGAGCA TCACCACAGA 2939 AGCCTCTCTG AGTTCACACT CCTCCTTGCT CCCTGCTCTG ACACAACAAT ACCCCATAT

Sequence length (SEQUENCE LENGTH): 878

Sequence type (SEQUENCE TYPE): amino acid

Sequence type (MOLECULE TYPE): Protein

Sequence (SEQUENCE DESCRIPTION):

 Met Asp Gin Gin Met Ala Leu Thr Trp Gly Leu Cys Tyr Met Ala Leu

 -20 -15 -10

 Val Ala Leu Cys Trp Gly His Glu Val Thr Glu Glu Glu Glu Thr Val

 -5 1 5 10

 Pro Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr Asn Arg lie He

 15 20 25

 Cys Ser Trp Ala Asp Thr Glu Asp Ala Gin Gly Leu He Asn Met Thr

 30 35 40

 Leu Leu Tyr His Gin Leu Asp Lys He Gin Ser Val Ser Cys Glu Leu

 45 50 55

 Ser Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val

 60 65 70

 Pro Arg Arg Cys Val lie Pro Tyr Thr Arg Phe Ser Asn Gly ASP Asn

 75 80 85 90

Asp Tyr Tyr Ser Phe Gin Pro Asp Arg Asp Leu Gly He Gin Leu Met 95 100 105

Val Pro Leu Ala Gin His Val Gin Pro Pro Pro Pro Lys Asp lie His

 110 115 120

 He Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu

 125 130 135

 Gly Asp Ser Gin Val Ser Trp Leu Ser Ser Lys Asp lie Glu Phe Glu

140 145 150

 Val Ala Tyr Lys Arg Leu Gin Asp Ser Trp Glu Asp Ala Ser Ser Leu 155 160 165 170

His Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys Leu Phe Leu Pro

 175 180 185

Asn Ser He Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser

 190 195 200

 Ser Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu Val His Trp Asp

 205 210 215

 Ser Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu Gin Cys Phe Phe

220 225 230

 Asp Gly lie Gin Ser Leu His Cys Ser Trp Glu Val Trp Thr Gin Thr 235 240 245 250

Thr Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro Ser Pro Ala Ala

 255 260 265

Pro Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val

 270 275 280

 Tyr Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu Pro Ser Ala His

 285 290 295

 Ser Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin Gly Lys Phe lie

300 305 310

Met Ser Tyr Tyr His lie Gin Met Glu Pro Pro He Leu Asn Gin Thr ^{5 9 o j d 3 "10} \ n UI9 asy OJ ^ naq u ΠΪΟ Jqi« S "10

 983 OSS 923

BIV ¾ OJJ J¾ Jiu dsv o ^j dv J9S OJd OJJ dsy Say θ] ΐ θ] ΐ usy

 02S gis oig

OJd dsy nio an ^η9 Ί OJJ jas \ usy asy dsv "10 neq STH nio

SOS 003 96 ^

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 0 09 na X | g sAq Xig dsv ui99Md naq ngq J9§ sXq J9S OJJ usy OJ ^

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 0

 9Md SIH nsi BIV ns naq naq J¾ aq 9Π nsq 91 ^ \ 2 naq an naq

S 0 9tf

 ΙΒΛ 9Π dJi naq jqi OJd ^ άιχ dsy Jill J¾ d jqj, J

0 QOi 'OO 96S usv dJi nig jas d an ΑϊΟ dsy i dsy J3S 811. ^J ds Ι¾Λ

 068 98S 08S

^{3J ΐ¾Λ 3JV BIV SAO J J9 S} J m dsv o j d "Ϊ3 UIO o j d Π9 Ί dsy

 S 8 OJLS 99S

 19N J3S usy SJ XI9 naq usy nig jqi sXq Jas dsy s q dj jas

098 9SS OSS nio J9S sAq sA sAq JAJ, UIO Ϊ¾Λ "10 ^ dms! H dsv 911 ^J s

 OK 9SS

 OJd ail sAq uio jqi djj, SIH jag i Jas dsy 3JV usy s

OSS SZS 028 918 OIS6d £ IJ d LLZ10I96O / A 540 545 550

lie Pro Ser Ser Arg Pro Arg Lys Gin Leu Pro Ser Phe Asp Phe Asn

 560 565 570

Gly Pro Tyr Leu Gly Pro Pro Gin Ser His Ser Leu Pro Asp Leu Pro

 575 580 585

Gly Gin Leu Gly Ser Pro Gin Val Gly Gly Ser Leu Lys Pro Ala Leu

 590 595 600

 Pro Gly Ser Leu Glu Tyr Met Cys Leu Pro Pro Gly Gly Gin Val Gin

 605 610 615

 Leu Val Pro Leu Ser Gin Val Met Gly Gin Gly Gin Ala Met Asp Val

620 625 630

 Gin Cys Gly Ser Ser Leu Glu Thr Thr Gly Ser Pro Ser Val Glu Pro 635 640 645 650

Lys Glu Asn Pro Pro Val Glu Leu Ser Val Glu Lys Gin Glu Ala Arg

 655 660 665

ASP Asn Pro Met Thr Leu Pro lie Ser Ser Gly Gly Pro Glu Gly Ser

 670 675 680

 Met Met Ala Ser Asp Tyr Val Thr Pro Gly Asp Pro Val Leu Thr Leu

 685 690 695

 Pro Thr Gly Pro Leu Ser Thr Ser Leu Gly Pro Ser Leu Gly Leu Pro

700 705 710

 Ser Ala Gin Ser Pro Ser Leu Cys Leu Lys Leu Pro Arg Val Pro Ser 715 720 725 730

Gly Ser Pro Ala Leu Gly Pro Pro Gly Phe Glu Asp Tyr Val Glu Leu

 735 740 745

Pro Pro Ser Val Ser Gin Ala Ala Thr Ser Pro Pro Gly His Pro Ala

 750 755 760

Pro Pro Val Ala Ser Ser Pro Thr Val lie Pro Gly Glu Pro Arg Glu 765 770 775

 Glu Val Gly Pro Ala Ser Pro His Pro Glu Gly Leu Leu Val Leu Arg

 780 785 790

 Gin Val Gly Asp Tyr Cys Phe Leu Pro Gly Leu Gly Pro Gly Ser Leu

795 800 805 810

 Ser Pro His Ser Lys Pro Pro Ser Pro Ser Leu Cys Ser Glu Thr Glu

 815 820 825

 Asp Leu Asp Gin Asp Leu Ser Val Lys Lys Phe Pro Tyr Gin Pro Leu

 830 835 840

 Pro Gin Ala Pro Al 〖le Gin Phe Phe Lys Ser Leu Lys Tyr

 845 850 855

SEQ ID NO: 8

Sequence length (SEQUENCE LENGTH): 1317

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): 2

 Topology (TOPOLOGY): linear

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 ATG GAC CAG CAA ATG GCA CTC ACA TGG GGG CTG TGC TAC ATG GCA CTG 48 Met Asp Gin Gin Met Ala Leu Thr Trp Gly Leu Cys Tyr Met Ala Leu

 -20 -15 -10

 GTG GCT CTC TGC TGG GGA CAC CAG GTG ACA GAG GAA GAA GAA ACG GTC 96 Val Ala Leu Cys Trp Gly His Glu Val Thr Glu Glu Glu Glu Thr Val

 -5 1 5 10

 CCT CTG AAG ACT CTG GAG TGC TAC AAT GAC TAC ACC AAC CGT ATC ATC 144 Pro Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr Asn Arg lie lie

 15 20 25

TGC AGC TGG GCA GAC ACA GAG GAT GCC CAG GGG CTA ATC AAC ATG ACC 192 Cys Ser Trp Ala Asp Thr Glu Asp Ala Gin Gly Leu lie Asn Met Thr

 30 35 40

 CTC CTC TAT CAC CAG CTA GAC AAG ATT CAA TCA GTG TCC TGT GAG CTC 240 Leu Leu Tyr His Gin Leu Asp Lys lie Gin Ser Val Ser Cys Glu Leu

 45 50 55

 AGT GAG AAA CTC ATG TGG TCA GAG TGC CCG TCA TCC CAC CGC TGT GTG 288 Ser Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val

 60 65 70

 CCC AGA AGA TGT GTC ATC CCC TAC ACA CGA TTT TCT AAT GGA GAT AAC 336 Pro Arg Arg Cys Val He Pro Tyr Thr Arg Phe Ser Asn Gly Asp Asn

 75 80 85 90

 GAC TAC TAC TCC TTC CAG CCA GAT CGT GAC CTG GGC ATC CAG CTC ATG 384 Asp Tyr Tyr Ser Phe Gin Pro Asp Arg Asp Leu Glylie Gin Leu Met

 95 100 105

 GTC CCA CTG GCC CAG CAT GTG CAG CCA CCC CCT CCC AAG GAC ATC CAC 432 Val Pro Leu Ala Gin His Val Gin Pro Pro Pro Pro Lys Asp lie His

 110 115 120

 ATC AGC CCC TCT GGG GAT CAT TTC CTG CTG GAA TGG AGT GTA TCT CTT 480 l ie Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu

 125 130 135

 GGG GAT TCC CAG GTC TCC TGG CTT TCA TCA AAG GAC ATA GAG TTT GAG 528 Gly Asp Ser Gin Val Ser Trp Leu Ser Ser Lys Asp lie Glu Phe Glu

 140 145 150

 GTG GCT TAT AAG CGG CTT CAG GAC TCC TGG GAG GAT GCC TCC AGT CTC 576 Val Ala Tyr Lys Arg Leu Gin Asp Ser Trp Giu Asp Ala Ser Ser Leu

155 160 165 170

CAC ACT AGC AAC TTT CAG GTG AAT TTA GAG CCA AAG CTA TTC CTA CCC 624 His Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys Leu Phe Leu Pro 175 180 185

AAC AGC ATC TAT GCT GCC CGT GTG CGC ACT CGG CTG TCC GCG GGT TCA 672 Asn Ser lie Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser

 190 195 200

 AGC TTG TCT GGG AGA CCC AGC AGA TGG AGC CCA GAG GTT CAC TGG GAC 720 Ser Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu Val His Trp Asp

 205 210 215

 TCC CAG CCA GGG GAC AAG GCC CAG CCA CAG AAC CTT CAA TGC TTC TTT 768 Ser Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu Gin Cys Phe Phe

 220 225 230

 GAT GGG ATC CAG TCT CTC CAC TGC TCC TGG GAG GTG TGG ACC CAG ACG 816 ASP Gly lie Gin Ser Leu His Cys Ser Trp Glu Val Trp Thr Gin Thr

235 240 245 250

 ACT GGC TCT GTT TCC TTT GGG CTC TTC TAT CGC CCC AGC CCT GCA GCT 864 Thr Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro Ser Pro Ala Ala

 255 260 265

 CCG GAG GAG AAA TGC TCT CCG GTG GTG AAG GAG CCG GAG GCC AGC GTC 912 Pro Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val

 270 275 280

 TAC ACC CGC TAC CGC TGC AGT CTA CCT GTG CCT GAG CCC AGT GCA CAC 960 Tyr Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu Pro Ser Ala His

 285 290 295

 AGC CAG TAC ACA GTC TCT GTT AAG CAC CTG GAA CAA GGG AAG TTC ATC 1008 Ser Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin Gly Lys Phe He

 300 305 310

 ATG AGC TAT TAC CAC ATC CAG ATG GAA CCT CCA ATC CTC AAC CAG ACC 1056 Met Ser Tyr Tyr His He Gin Met Glu Pro Pro lie Leu Asn Gin Thr

315 320 325 330 AAG AAC AGA GAC AGC TAC AGC CTG CAT TGG GAA ACT CAG AAA ATA CCC 1104 Lys Asn Arg Asp Ser Tyr Ser Leu His Trp Glu Thr Gin Lys He Pro

 335 340 345

 AAA TAC ATT GAT CAC ACT TTC CAG GTC CAG TAC AAG AAG AAG TCA GAG 1152 Lys Tyr lie Asp His Thr Phe Gin Val Gin Tyr Lys Lys Lys Ser Glu

 350 355 360

 AGC TGG AAG GAC AGC AAG ACA GAA AAC CTA GGT CGA GTC AAT AGC ATG 1200 Ser Trp Lys Asp Ser Lys Thr Glu Asn Leu Gly Arg Val Asn Ser Met

 365 370 375

 GAC CTG CCC CAG CTG GAG CCG GAC ACC TCA TAC TGC GCC AGG GTG AGG 1248 Asp Leu Pro Gin Leu Glu Pro Asp Thr Ser Tyr Cys Ala Arg Val Arg

 380 385 390

GTC AAG CCC ATC TCT GAC TAC GAC GGG ATC TGG AGC GAG TGG AGC AAT 1296 Val Lys Pro lie Ser Asp Tyr Asp Gly He Trp Ser Glu Trp Ser Asn

 395 400 405 410

GAG TAC ACT TGG ACG ACT GAC 1317 Glu Tyr Thr Trp Thr Thr Asp

 415

SEQ ID NO: 9

Sequence length (SEQUENCE LENGTH): 439

Sequence type (SEQUENCE TYPE): amino acid

Sequence type (MOLECULE TYPE): Protein

Sequence (SEQUENCE DESCRIPTION):

 Met Asp Gin Gin Met Ala Leu Thr Trp Cly Leu Cys Tyr Met Ala Leu

 -20 -15 -10

 Val Ala Leu Cys Trp Cly His Glu Val Thr Glu Glu Glu Glu Thr Val

 -5 1 5 10

Pro Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr Asn Arg lie He 15 20 25

Cys Ser Trp Ala AS P Thr Glu Asp Ala Gin Gly Leu He Asn Met Thr

 30 35 40

 Leu Leu Tyr His Gin Leu Asp Lys lie Gin Ser Val Ser Cys Glu Leu

 45 50 55

 Ser Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val

60 65 70

 Pro Arg Arg Cys Val lie Pro Tyr Thr Arg Phe Ser Asn Gly Asp Asn 75 80 85 90

Asp Tyr Tyr Ser Phe Gin Pro ASP Arg Asp Leu Gly 〖k Gin Leu Met

 95 100 105

Val Pro Leu Ala Gin His Val Gin Pro Pro Pro Pro Lys ASP lie His

 110 115 120

[L e Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu

 125 130 135

 Gly Asp Ser Gin Val Ser Trp Leu Ser Ser Lys Asp He Glu Phe Glu

140 145 150

 Val Ala Tyr Lys Arg Leu Gin Asp Ser Trp Glu Asp Ala Ser Ser Leu 155 160 165 170

His Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys Leu Phe Leu Pro

 175 180 185

Asn Ser He Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser

 190 195 200

 Ser Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu Val His Trp Asp

 205 210 215

 Ser Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu Gin Cys Phe Phe

220 225 230

Asp Gly lie Gin Ser Leu His Cys Ser Trp Glu Val Trp Thr Gin Thr 235 240 245 250

Thr Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro Ser Pro Ala Ala

 255 260 265

Pro Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val

 270 275 280

 Tyr Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu Pro Ser Ala His

 285 290 295

 Ser Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin Gly Lys Phe lie

300 305 310

 Met Ser Tyr Tyr His lie Gin Met Glu Pro Pro 〖le Leu Asn Gin Thr 315 320 325 330

Lys Asn Arg ASP Ser Tyr Ser Leu His Trp Glu Thr Gin Lys lie Pro

 335 340 345

Lys Tyr He Asp His Thr Thr Phe Gin Val Gin Tyr Lys Lys Lys Ser Glu

 350 355 360

 Ser Trp Lys Asp Ser Lys Thr Glu Asn Leu Gly Arg Val Asn Ser Met

 365 370 375

 Asp Leu Pro Gin Leu Glu Pro Asp Thr Ser Tyr Cys Ala Arg Val Arg

380 385 390

 Val Lys Pro 〖le Ser Asp Tyr Asp Gly He Trp Ser Glu Trp Ser Asn 395 400 405 410

Glu Tyr Thr Trp Thr Thr Asp

 415

SEQ ID NO: 10

Sequence length (SEQUENCE LENGTH): 1800

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): 2

Topology (T0P0 then 0GY): linear Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 ATG CTG GGC ATC TGG ACC CTC CTA CCT CTG GTT CTT ACG TCT GTT GCT 48 Met Leu Gly lie Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala

 -15 -10 -5

 AGA TTA TCG TCC AAA ACT GTT AAT GCC CAA GTG ACT GAC ATC AAC TCC 96 Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp He Asn Ser

 1 5 10 15

 AAG GGA TTG GAA TTG AGG AAG ACT GTT ACT ACA GTT GAG ACT CAG AAC 144 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn

 20 25 30

 TTC GAA GGC CTG CAT CAT GAT CCC CAA TTC TGC CAT AAG CCC TGT CCT 192 Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro

 35 40 45

 CCA GGT GAA AGG AAA GCT AGG GAC TGC ACA GTC AAT GGG GAT GAA CCA 240 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro

 50 55 60

 GAC TGC GTG CCC TGC CAA GAA GGG AAG GAG TAC ACA GAC AAA CCC CAT 288 Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His

 65 70 75 80

 TTT TCT TCC AAA TGC AGA AGA TGT AGA TTG TGT GAT GAA GGA CAT GGC 336 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly

 85 90 95

 TTA GAA GTG GAA ATA AAC TGC ACC CGG ACC CAG AAT ACC AAG TCC AGA 384 Leu Glu Val Glu He Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg

 100 105 110

TGT AAA CCA AAC TTT TTT TGT AAC TCT ACT GTA TGT GAA CAC TGT GAC 432 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 115 120 125

 CCT TGC ACC AAA TGT GAA CAT GGA ATC ATC AAG GAA TGC ACA CTC ACC 480 Pro Cys Thr Lys Cys Glu His Gly lie lys Lys Glu Cys Thr Leu Thr

 130 135 140

 AGC AAC ACC AAG TGC AAA GAG GAA GGA TCC AGA TCG GTC GCG GCC GTG 528 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Val Ala Ala Val

145 150 155 160

 ACA GAG GAA GAA GAA ACG GTC CCT CTG AAG ACT CTG GAG TGC TAC AAT 576 Thr Glu Glu Glu Glu Thr Val Pro Leu Lys Thr Leu Glu Cys Tyr Asn

 165 170 175

 GAC TAC ACC AAC CGT ATC ATC TGC AGC TGG GCA GAC ACA GAG GAT GCC 624 Asp Tyr Thr Asn Arg lie 〖le Cys Ser Trp Ala Asp Thr Glu Asp Ala

 180 185 190

 CAG GGG CTA ATC AAC ATG ACC CTC CTC TAT CAC CAG CTA GAC AAG ATT 672 Gin Gly Leu lie Asn Met Thr Leu Leu Tyr His Gin Leu Asp Lys lie

 195 200 205

 CAA TCA GTG TCC TGT GAG CTC AGT GAG AAA CTC ATG TGG TCA GAG TGC 720 Gin Ser Val Ser Cys Glu Leu Ser Glu Lys Leu Met Trp Ser Glu Cys

210 215 ¹ 220

 CCG TCA TCC CAC CGC TGT GTG CCC AGA AGA TGT CTC ATC CCC TAC ACA 768 Pro Ser Ser His Arg Cys Val Pro Arg Arg Cys Val lie Pro Tyr Thr

225 230 235 240

 CGA TTT TCT AAT GGA GAT AAC GAC TAC TAC TCC TTC CAG CCA GAT CGT 816 Arg Phe Ser Asn Gly Asp Asn Asp Tyr Tyr Ser Phe Gin Pro Asp Arg

 245 250 255

 GAC CTG GGC ATC CAG CTC ATG GTC CCA CTG GCC CAG CAT GTG CAG CCA 864 Asp Leu Gly lie Gin Leu Met Val Pro Leu Ala Gin His Val Gin Pro

260 265 270 CCC CCT CCC AAG GAC ATC CAC ATC AGC CCC TCT GGG GAT CAT TTC CTG 912 Pro Pro Pro Lys Asp lie His lie Ser Pro Ser Gly Asp His Phe Leu

 275 280 285

 CTG GAA TGG ACT GTA TCT CTT GGG GAT TCC CAG GTC TCC TGG CTT TCA 960 Leu Glu Trp Ser Val Ser Leu Gly Asp Ser Gin Val Ser Trp Leu Ser

 290 295 300

 TCA AAG GAC ATA GAG TTT GAG GTG GCT TAT AAG CGG CTT CAG GAC TCC 1008 Ser Lys Asp 〖le Glu Phe Glu Val Ala Tyr Lys Arg Leu Gin Asp Ser

305 310 315 320

 TGG GAG GAT GCC TCC ACT CTC CAC ACT AGC AAC TTT CAG GTG AAT TTA 1056 Trp Glu Asp Ala Ser Ser Leu His Thr Ser Asn Phe Gin Val Asn Leu

 325 330 335

 GAG CCA AAG CTA TTC CTA CCC AAC AGC ATC TAT GCT GCC CGT GTG CGC 1104 Glu Pro Lys Leu Phe Leu Pro Asn Ser He Tyr Ala Ala Arg Val Arg

 340 345 350

 ACT CGG CTG TCC GCG GGT TCA AGC TTG TCT GGG AGA CCC AGC AGA TGG 1152 Thr Arg Leu Ser Ala Gly Ser Ser Leu Ser Gly Arg Pro Ser Arg Trp

 355 360 365

 AGC CCA GAG GTT CAC TGG GAC TCC CAG CCA GGG GAC AAG GCC CAG CCA 1200 Ser Pro Glu Val His Trp Asp Ser Gin Pro Gly Asp Lys Ala Gin Pro

 370 375 380

 CAG AAC CTT CAA TGC TTC TTT GAT GGG ATC CAG TCT CTC CAC TGC TCC 1248 Gin Asn Leu Gin Cys Phe Phe Asp Glylie Gin Ser Leu His Cys Ser

385 390 395 400

 TGG GAG GTG TGG ACC CAG ACG ACT GGC TCT GTT TCC TTT GGG CTC TTC 1296 Trp Glu Val Trp Thr Gin Thr Thr Gly Ser Val Ser Phe Gly Leu Phe

 405 410 415

TAT CGC CCC AGC CCT GCA GCT CCG GAG GAG AAA TGC TCT CCG GTG GTG 1344 Tyr Arg Pro Ser Pro Ala Ala Pro Glu Glu Lys Cys Ser Pro Val Val

 420 425 430

 AAG GAG CCG GAG GCC AGC GTC TAC ACC CGC TAC CGC TGC ACT CTA CCT 1392 Lys Glu Pro Gin Ala Ser Val Tyr Thr Arg Tyr Arg Cys Ser Leu Pro

 435 440 445

 GTG CCT GAG CCC AGT GCA CAC AGC CAG TAC ACA GTC TCT GTT AAG CAC 1440 Val Pro Glu Pro Ser Ala His Ser Gin Tyr Thr Val Ser Val Lys His

 450 455 460

 CTG GAA CAA GGG AAG TTC ATC ATG AGC TAT TAC CAC ATC CAG ATG GAA 1488 Leu Glu Gin Gly Lys Phe lie Met Ser Tyr Tyr His lie Gin Met Glu

465 470 475 480

 CCT CCA ATC CTC AAC CAG ACC AAG AAC AGA GAC AGC TAC AGC CTG CAT 1536 Pro Pro lie Leu Asn Gin Thr ys Asn Arg Asp Ser Tyr Ser Leu His

 485 490 495

 TGG GAA ACT CAG AAA ATA CCC AAA TAC ATT GAT CAC ACT TTC CAG GTC 1584 Trp Glu Thr Gin Lys lie Pro Lys Tyr lie Asp His Thr Phe Gin Val

 500 505 510

 CAG TAC AAG AAG AAG TCA GAG AGC TGG AAG GAC AGC AAG ACA GAA AAC 1632 Gin Tyr Lys Lys Lys Ser Glu Ser Trp Lys Asp Ser Lys Thr Glu Asn

 515 520 525

 CTA GGT CGA GTC AAT AGC ATG GAC CTG CCC CAG CTG GAG CCG GAC ACC 1680 Leu Gly Arg Val Asn Ser Met Asp Leu Pro Gin Leu Glu Pro Asp Thr

 530 535 540

 TCA TAC TGC GCC AGG GTG AGG GTC AAG CCC ATC TCT GAC TAC GAC GGG 1728 Ser Tyr Cys Ala Arg Val Arg Val Lys Pro lie Ser Asp Tyr Asp Gly

545 550 555 560

ATC TGG AGC GAG TGG AGC AAT GAG TAC ACT TGG ACG ACT GAC TGG GTG 1776 l ie Trp Ser Glu Trp Ser Asn Glu Tyr Thr Trp Thr Thr Asp Trp Val 565 570 575

ATG CCC ACG GCC GCT AGA CTA GTC 1800 Met Pro Thr Ala Ala Arg Leu Val

 580

SEQ ID NO: 11

Sequence length (SEQUENCE LENGTH): 584

Sequence type (SEQUENCE TYPE) '· amino acid

Sequence type (MOLECULE TYPE): Protein

Sequence (SEQUENCE DESCRIPTION):

 Arg Leu Ser Ser Lys Ser Val Asn Ala Gin Val Thr Asp lie Asn Ser

 1 5 10 15

 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn

 20 25 30

 Leu Glu Gly Leu His His Asp Gly Gin Phe Cys His Lys Pro Cys Pro

 35 40 45

 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro

 50 55 60

 Asp Cys Val Pro Cys Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His

 65 70 75 80

 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly

 85 90 95

 Leu Glu Val Glu lie Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg

 100 105 110

 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp

 115 120 125

 Pro Cys Thr Lys Cys Glu His Gly lie lye Lys Glu Cys Thr Leu Thr

 130 135 140

Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Val Ala Ala Val 145 150 155 160

Thr Glu Glu Glu Glu Thr Val Pro Leu Lys Thr Leu Glu Cys Tyr Asn

 165 170 175

Asp Tyr Thr Asn Arg 〖le lie Cys Ser Trp Ala Asp Thr Glu Asp Ala

 180 185 190

 Gin Gly Leu lie Asn Met Thr Leu Leu Tyr His Gin Leu Asp Lys lie

 195 200 205

 Gin Ser Val Ser Cys Glu Leu Ser Glu Lys Leu Met Trp Ser Glu Cys

210 215 220

 Pro Ser Ser His Arg Cys Val Pro Arg Arg Cys Val lie Pro Tyr Thr 225 230 235 240

Arg Phe Ser Asn Gly Asp Asn Asp Tyr Tyr Ser Phe Gin Pro Asp Arg

 245 250 255

Asp Leu Gly He Gin Leu Met Val Pro Leu Ala Gin His Val Gin Pro

 260 265 270

 Pro Pro Pro Lys Asp lie His lie Ser Pro Ser Gly Asp His Phe Leu

 275 280 285

 Leu Glu Trp Ser Val Ser Leu Gly Asp Ser Gin Val Ser Trp Leu Ser

290 295 300

 Ser Lys Asp He Glu Phe Glu Val Ala Tyr Lys Arg Leu Gin Asp Ser 305 310 315 320

Trp Glu Asp Ala Ser Ser Leu His Thr Ser Asn Phe Gin Val Asn Leu

 325 330 335

Glu Pro Lys Leu Phe Leu Pro Asn Ser He Tyr Ala Ala Arg Val Arg

 340 345 350

 Thr Arg Leu Ser Ala Gly Ser Ser Leu Ser Gly Arg Pro Ser Arg Trp

 355 360 365

Ser Pro Glu Val His Trp Asp Ser Gin Pro Gly Asp Lys Ala Gin Pro 370 375 380

 Gin Asn Leu Gin Cys Phe Phe Asp Gly lie Gin Ser Leu His Cys Ser 385 390 395 400

Trp Glu Val Trp Thr Gin Thr Thr Gly Ser Val Ser Phe Gly Leu Phe

 405 410 415

Tyr Arg Pro Ser Pro Ala Ala Pro Glu Glu Lys Cys Ser Pro Val Val

 420 425 430

 Lys Glu Pro Gin Ala Ser Val Tyr Thr Arg Tyr Arg Cys Ser Leu Pro

 435 440 445

 Val Pro Glu Pro Ser Ala His Ser Gin Tyr Thr Val Ser Val Lys His

450 455 460

 Leu Glu Gin Gly Lys Phe lie Met Ser Tyr Tyr His lie Gin Met Glu 465 470 475 480

Pro Pro lie Leu Asn Gin Thr Lys Asn Arg Asp Ser Tyr Ser Leu His

 485 490 495

Trp Glu Thr Gin Lys lie Pro Lys Tyr lie Asp His Thr Phe Gin Val

 500 505 510

 Gin Tyr Lys Lys Lys Ser Glu Ser Trp Lys Asp Ser Lys Thr Glu Asn

 515 520 525

 Leu Gly Arg Val Asn Ser Met Asp Leu Pro Gin Leu Glu Pro Asp Thr

530 535 540

 Ser Tyr Cys Ala Arg Val Arg Val Lys Pro lie Ser As Tyr Asp Gly 545 550 555 560 lie Trp. Ser Glu Trp Ser Asn Glu Tyr Thr Trp Thr Thr Asp Trp Val

 565 570 575

Met Pro Thr Ala Ala Arg Leu Val

 580

SEQ ID NO: 12

7 g Sequence length (SEQUENCE LENGTH): 1506

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

 Topology: TOPOLOGY

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 GCCGCAGGCT GCCCACACAG GCCGCCCGCT GTTTTCCCTT GCTGCAGAC ATG CTG TGG 58

 Met Leu Trp

 -20

 ATC TGG GCT GTC CTG CCT CTG GTG CTT GCT GGC TCA CAG TTA AGA GTT 106 lie Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gin Leu Arg Val

 -15 -10 -5

 CAT ACT CAA GGT AGT AAT AGC ATC TCC GAG AGT TTA AAG CTG AGG AGG 154 His Thr Gin Gly Thr Asn Ser He Ser Glu Ser Leu Lys Leu Arg Arg

 1 5 10

 CGG GTT CAT GAA ACT GAT AAA AAC TGC TCA GAA GGA TTA TAT CAA GGA 202 Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu Tyr Gin Gly

 15 20 25 30

 GGC CCA TTT TGC TGT CAA CCA TGC CAA CCT GGT AAA AAA AAA GTT GAG 250 Gly Pro Phe Cys Cys Gin Pro Cys Gin Pro Gly Lys Lys Lys Val Glu

 35 40 45

 GAC TGC AAA ATG AAT GGG GGT ACA CCA ACC TGT GCC CCA TGC ACA GAA 298 Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro Cys Thr Glu

 50 55 60

 GGG AAG GAG TAC ATG GAC AAG AAC CAT TAT GCT GAT AAA TGC AGA AGA 346 Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys Cys Arg Arg

 65 70 75

 TGC ACA CTC TGC GAT GAA GAG CAT GGT TTA GAA GTG GAA ACA AAC TGC 394

8 o Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu Thr Asn Cys

 80 85 90

 ACC CTG ACC CAG AAT ACC AAG TGC AAG TGC AAA CCA GAC TTC TAC TGC 442 Thr Leu Thr Gin Asn Thr Lys Cys Lys Cys Lys Pro Asp Phe Tyr Cys

 95 100 105 110

 GAT TCT CCT GGC TGT GAA CAC TGT GTT CGC TGC GCC TCG TGT GAA CAT 490 ASP Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser Cys Glu His

 115 120 125

 GGA ACC CTT GAG CCA TGC ACA GCA ACC AGC AAT ACA AAC TGC AGG AAA 538 Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn Cys Arg Lys

 130 135 140

 CAA AGT CCC AGA AAT CGC CTA TCG TTG TTG ACC ATC CTT GTT TTG TTA 586 Gin Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr He He Leu Val Leu Leu

 145 150 155

 ATT CCA CTT GTA TTT ATA TAT CGA AAG TAC CGG AAA AGA AAG TGC TGG 634 lie Pro Leu Val Phe lie Tyr Arg Lys Tyr Arg Lys Arg Lys Cys Trp

 160 165 170

 AAA AGG AGA CAG GAT GAC CCT GAA TCT AGA ACC TCC AGT CGT GAA ACC 682 Lys Arg Arg Gin Asp Asp Pro Glu Ser Arg Thr Ser Ser Arg Glu Thr

175 180 185 190

 ATA CCA ATG AAT GCC TCA AAT CTT AGC TTG AGT AAA TAC ATC CCG AGA 730 l ie Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr lie Pro Arg

 195 200 205

 ATT GCT GAA GAC ATG ACA ATC CAG GAA GCT AAA AAA TTT GCT CGA GAA 778 l ie Ala Glu Asp Met Thr He Gin Glu Ala Lys Lys Phe Ala Arg Glu

 210 215 220

 AAT AAC ATC AAG GAG GGC AAG ATA GAT GAG ATC ATG CAT GAC AGC ATC 826 Asn Asn He Lys Glu Gly Lys lie Asp Glu lie Met His ASP Ser lie

8 l 225 230 235

 CAA GAC ACA GCT GAG CAG AAA GTC CAG CTG CTC CTG TGC TGG TAC CAA 874 Gin Asp Thr Ala Glu Gin Lys Val Gin Leu Leu Leu Cys Trp Tyr Gin

 240 245 250

 TCT CAT GGG AAG AGT GAT GCA TAT CAA GAT TTA ATC AAG GGT CTC AAA 922 Ser His Gly Lys Ser Asp Ala Tyr Gin Asp Leu ly Lys Gly Leu Lys

255 260 265 270

 AAA GCC GAA TGT CGC AGA ACC TTA GAT AAA TTT CAG GAC ATG GTC CAG 970 Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gin Asp Met Val Gin

 275 280 285

 AAG GAC CTT GGA AAA TCA ACC CCA GAC ACT GGA AAT GAA AAT GAA GGA 1018 Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu Asn Glu Gly

 290 295 300

 CAA TGT CTG GAG TGAAAACTAC CTCAGTTCCA GCCATGAAGA GAGGAGAGAG 1070 Gin Cys Leu Glu

 305

 CCTGCCACCC ATGATGGAAA CAAAATGAAT GCCAACTGTA TTGACATTGG CAACTCCTGG 1130

TGTGTTCTCT TTGCCAGCAA ATGGTAGTTG ATACTCAGTG AGGGTCAAAT GACTAGCAGG 1190

TTCCAGGGAC TGCTTCTGTT ATTCTCTGCA GTTGCTGAGA TGAACCATTT TCTCTGTCTA 1250

CTGCAATTTT TACATTCAAA TGTCCATGAA ATTTGTATTA AATGTGAAGT GGAATCTGCA 1310

GTCTTTGTGT TTATATTCAT ATACTATGAA CTGAGGAGAA TTATAAACTG AAACAAATAC 1370

TCGCAGTTAA TTGAAGACCT TCCATTGATG GACAGTTCTT TTCCTCTCTA TATGGAAATG 1430

TATAATACAA GAAATAATTT TTAAATTAAA GTATCTCTTT TTGCATTTCA AAAAAAAAAA 1490

AAAAAAAAAA AAAAAA 1506 SEQ ID NO: 13

Sequence length (SEQUENCE LENGTH): 327

Sequence type (SEQUENCE TYPE): amino acid

Sequence type (MOLECULE TYPE): Protein Array (SEQUENCE DESCRIPTION):

 Met Leu Trp lie Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gin

-20 -15 -10

 Leu Arg Val His Thr Gin Gly Thr Asn Ser lie Ser Glu Ser Leu Lys -5 1 5 10

Leu Arg Arg Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu

 15 20 25

 Tyr Gin Gly Gly Pro Phe Cys Cys Gin Pro Cys Gin Pro Gly Lys Lys

 30 35 40

 Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro

45 50 55

 Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys 60 65 70 75

Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu

 80 85 90

Thr Asn Cys Thr Leu Thr Gin Asn Thr Lys Cys Lys Cys Lys Pro Asp

 95 100 105

 Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser

 110 115 120

 Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn

125 130 135

 Cys Arg Lys Gin Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr lie Leu 140 145 150 155

Val Leu Leu He Pro eu Val Phe lie Tyr Arg Lys Tyr Arg Lys Arg

 160 165 170

Lys Cys Trp Lys Arg Arg Gin Asp Asp Pro Glu Ser Arg Thr Ser Ser

 175 180 185

Arg Glu Thr lie Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr 190 195 200

l ie Pro Arg lie Ala Glu Asp Met Thr l ie Gin Glu Ala Lys Lys Phe

 205 210 215

 Ala Arg Glu Asn Asn lie Lys Glu Gly Lys lie Asp Glu He Met His

220 225 230 235

 Asp Ser lie Gin Asp Thr Ala Glu Gin Lys Val Gin Leu Leu Leu Cys

 240 245 250

 Trp Tyr Gin Ser His Gly Lys Ser Asp Ala Tyr Gin Asp Leu lie Lys

 255 260 265

 Gly Leu Lys Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gin Asp

 270 275 280

 Met Val Gin Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu

 285 290 295

 Asn Glu Gly Gin Cys Leu Glu

300 305

SEQ ID NO: 14

Sequence length (SEQUENCE LENGTH): 20

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

 Topology 1 (TOPOLOGY): Straight down

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 TATGGCACGA GGTGACGCGT 20 SEQ ID NO: 15

Sequence length (SEQUENCE LENGTH): 20

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

Topology (TOPOLOGY): linear Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 CGTGCTCCAC TGCGCAGATC 20 SEQ ID NO: 16

Sequence length (SEQUENCE LENGTH): 507

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

 Topology (TOPOLOGY): linear

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 ATG CTG TGG ATC TGG GCT GTC CTG CCT CTG GTG CTT GCT GGC TCA CAG 48 Met Leu Trp lie Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gin

 -20 -15 -10

 TTA AGA GTT CAT ACT CAA GGT AGT AAT AGC ATC TCC GAG AGT TTA AAG 96 Leu Arg Val His Thr Gin Gly Thr Asn Ser lie Ser Glu Ser Leu Lys

 -5 1 5 10

 CTG AGG AGG CGG GTT CAT GAA ACT GAT AAA AAC TGC TCA GAA GGA TTA 144 Leu Arg Arg Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu

 15 20 25

 TAT CAA GGA GGC CCA TTT TGC TGT CAA CCA TGC CAA CCT GGT AAA AAA 192 Tyr Gin Gly Gly Pro Phe Cys Cys Gin Pro Cys Gin Pro Gly Lys Lys

 30 35 40

 AAA GTT GAG GAC TGC AAA ATG AAT GGG GGT ACA CCA ACC TGT GCC CCA 240 Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro

 45 50 55

 TGC ACA GAA GGG AAG GAG TAC ATG GAC AAG AAC CAT TAT GCT GAT AAA 288 Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys

60 65 70 75 9 8

Ot '98 OS

 s sA i9 old uio SAQ. «10 s 9Md oJd λι Λΐ9« ΐθ

S3 02 SI na Aio nio J8S SAO usy sA dsy J¾ n | g STH Ι¾Λ V 3-iV 3 ^J V nsq

01 9 ΐ 9- sAq Π9ΐ J3S nio J9 $ ΘΠ JQS usy jqi Λ19 ui ^Jl U siH 3iV nsq

 01- 91- 03-

«Ϊ0 ^J 9S 3 BIV nai Ι¾Λ ns O ^ ngq Ι¾Λ ¾IV dJJL d \\ dj 丄 ng jgjy

 : (NOUdiaOSaa 33N3ilD3S) M3M 驩, OdAl aO 3n53S) filCi? Ii 691: (H10N3133N3nb3S) ^^ OfiiSM

LI ^: (ON ai 63S) "" 銎 『31

 on

3JV usy 3JV OJd J9S uio s 8JV SAQ9 093 IVV V9V 03 310V VV3 VVV 09V 301

 381 OSI 921 usv m usv s jqx BIV J¾ SAQ OJJ nio nsq J¾ Aig SIH nt SAQ

OVV VOV IVV 30V 00V V30 V3V 001 VOO 9V911033V VOO IVO WO 131

 021 gn 011

 J9S ¾IV SAO 3JV ϊ¾Λ SAO STH nig SAQ A OJd J9S dsy SXQ UI aqj

001030001 D031101313V3 WO 101003130101 IVO 091 OVl Oil

 901 001 96 dsy o j SAq SAQ sAq SAQ s jqi usy uig jqi ng J¾ s usy ¾

OVO VOO VVV 301 OVV 001 OVV 33 IVV 9V3 33V 013 33V 031 OVV V3V

06 S8 08

nio Λ nio ⁿ 3 SIR nig nig dsy SAQ na J¾ s 3jy 3JV sS WO 019 VV9 Vll 100 IVO 0V9 WO IVO 031310 VOV 391 V9V VOV 001 £ 00IS6d £ iIDd LLZW96 OA Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro

 45 50 55

 Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys

 60 65 70 75

 Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu

 80 85 90

 Thr Asn Cys Thr Leu Thr Gin Asn Thr Lys Cys Lys Cys Lys Pro Asp

 95 100 105

 Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser

 110 115 120

 Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn

 125 130 135

 Cys Arg Lys Gin Ser Pro Arg Asn Arg

140 145

SEQ ID NO: 18

Sequence length (SEQUENCE LENGTH): 1776

Sequence type (SEQUENCE TYPE): nucleic acid

Number of chains (STRANDNESS): Double strand

 Topology— (TOPOLOGY): Linear

Sequence type (MOLECULE TYPE): cDNA

Sequence (SEQUENCE DESCRIPTION):

 ATG CTG TGG ATC TGG GCT GTC CTG CCT CTG GTG CTT GCT GGC TCA CAG 48 Met Leu Trp He Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gin

 -20 -15 -10

 TTA AGA GTT CAT ACT CAA GGT ACT AAT AGC ATC TCC GAG ACT TTA AAG 96 Leu Arg Val His Thr Gin Gly Thr Asn Ser lie Ser Glu Ser Leu Lys

 -5 1 5 10

CTG AGG AGG CGG GTT CAT GAA ACT GAT AAA AAC TGC TCA GAA GGA TTA 144 Eu Arg Arg Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu

 15 20 25

 TAT CAA CGA GGC CCA TTT TGC TGT CAA CCA TGC CAA CCT GGT AAA AAA 192 Tyr Gin Gly Gly Pro Phe Cys Cys Gin Pro Cys Gin Pro Gly Lys Lys

 30 35 40

 AAA GTT CAG GAC TGC AAA ATG AAT GGG GGT ACA CCA ACC TGT GCC CCA 240 Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro

 45 50 55

 TGC ACA GAA GGG AAG GAG TAC ATG GAC AAG AAC CAT TAT GCT GAT AAA 288 Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys

 60 65 70 75

 TGC AGA AGA TGC ACA CTC TGC GAT GAA GAG CAT GGT TTA GAA GTG GAA 336 Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu

 80 85 90

 ACA AAC TGC ACC CTC ACC CAG AAT ACC AAG TGC AAG TGC AAA CCA GAC 384 Thr Asn Cys Thr Leu Thr Gin Asn Thr Lys Cys Lys Cys Lys Pro Asp

 95 100 105

 TTC TAC TGC GAT TCT CCT GGC TGT GAA CAC TGT GTT CGC TGC GCC TCG 432 Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser

 110 115 120

 TGT GAA CAT GGA ACC CTT CAG CCA TGC ACA GCA ACC AGC AAT ACA AAC 480 Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn

 125 130 135

TGC AGG AAA CAA AGT CCC AGA AAT CGC CTA TGG CAC GTG ACA GAG CAA 528 Cys Arg Lys Gin Ser Pro Arg Asn Arg Leu Trp His Val Thr Glu Glu

140 145 150 155

GAA GAA ACG GTC CCT CTC AAG ACT CTG GAG TGC TAC AAT GAC TAC ACC 576 Glu Glu Thr Val Pro Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr 6 8

SIS 018 90S 00S dsv "10 ^d « U s dsy «10 naq 3JV sAq BIV Λ nio aqd nio 811 00 Ϊ IVO OVO 001 301 OVO OVO 113 000 3VV XVI 100 010 OVO 111 3V0 VIV

963 06Z 98Z dsv sAq J9S i3S ng dJi 35 ato ¹⁹ S dsy A13 Π9 J9s Ι¾Λ J ^ S 096 OVG OVV V01 V01 110 001 301 019 0V3 331 1V9 900 110 XOX VIO IOV

 083 giZ QLZ.

dJi n ndi na gqj S IH dsy Aio s OJJ JSS d \ S IH 81 1 DS V sAq 216 091 WO 013 313 311 1V3 IVO 009 131 000 OOV OIV OVO 31V OVO OVV

 99Z 093 992

 OJd o d OJd OJd uio ΐ¾Λ S IH UTO ¾TV naq 0 Ι¾Λ «10 91 1 m 000 103 003 VOO 9V3 310 1V3 3V3 DOO 010 V33 010 91V 310 0V3 OIV

 093 m m

 AIO na dsv 3JV dsv oid uig aqj jas JAX iki dsy usy dsv A19 usy 918 309 910 0V9 193 IVO VOO OVO Oil 001 OVl OVl OVO OVV IVO VOO IVV

 983 082 S3Z 022

J9S 9Md 3JV JMl Ul OJd si] in SAQ 8JV 3JV 0 SA3 3JV SiH 89 131 111 VOO VOV 3V1 000 31V OID 131 VOV V3V 030 919 101 003 OVO

^{912 012 303 i9S S o J d} sAo "to dJ JL Η3 Ί sA n jas naq n SAQ S ZL 331 VOX OOD 001 0V9 VOX 091 OIV 010 VVV OVO IOV 310 3V0 101 031

 OOZ 961 061 m J9S UIO ai l sAq dsv nsi uio S IH i ngq na jqi i9j¾ usv ail

ZL2 310 V31 m HV OVV 3V3 VIO OVO OVO 1V1 013 010 33V 31V OVV 3XV

 281 08 ϊ 92.1

 nsq Aio U13 ¾iv dsy J¾ dsy ¾ΐν dJi sXQ 1 9 Π 3JV usy 9 VIO 900 OVO 033 IVO OVO VOV 0V9 V33 001 03V 091 OIV OIV 100 OVV

 OLl 99 t 09 ΐ

6f £ 00 / S6d £ / ld LL110I96 O / A GCC TCC AGT CTC CAC ACT AGC AAC TTT CAG GTG AAT TTA GAG CCA AAG 1056 Ala Ser Ser Leu His Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys

 320 325 330

 CTA TTC CTA CCC AAC AGC ATC TAT GCT GCC CGT GTG CGC ACT CGG CTG 1104 Leu Phe Leu Pro Asn Ser Ile Tyr Ala Ala Arg Val Arg Thr Arg Leu

 335 340 345

 TCC GCG GGT TCA AGC TTG TCT GGG AGA CCC AGC AGA TGG AGC CCA GAG 1152 Ser Ala Gly Ser Ser Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu

 350 355 360

 GTT CAC TGG GAC TCC CAG CCA GGG GAC AAG GCC CAG CCA CAG AAC CTT 1200 Val His Trp Asp Ser Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu

 365 370 375

 CAA TGC TTC TTT GAT GGG ATC CAG TCT CTC CAC TGC TCC TGG GAG GTG 1248 Gin Cys Phe Phe Asp Gly He Gin Ser Leu His Cys Ser Trp Glu Val

380 385 390 395

 TGG ACC CAG ACG ACT GGC TCT GTT TCC TTT GGG CTC TTC TAT CGC CCC 1296 Trp Thr Gin Thr Thr Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro

 400 405 410

 AGC CCT GCA GCT CCG GAG GAG AAA TGC TCT CCG GTG GTG AAG GAG CCG 1344 Ser Pro Ala Ala Pro Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro

 415 420 425

 GAG GCC AGC GTC TAC ACC CGC TAC CGC TGC AGT CTA CCT GTG CCT GAG 1392 Gin Ala Ser Val Tyr Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu

 430 435 440

 CCC AGT GCA CAC AGC CAG TAC ACA GTC TCT CTT AAG CAC CTG GAA CAA 1440 Pro Ser Ala His Ser Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin

 445 450 455

GGG AAG TTC ATC ATG AGC TAT TAC CAC ATC CAG ATG GAA CCT CCA ATC 1488 Gly Lys Phe 〖le Met Ser Tyr Tyr His lie Gin Met Glu Pro Pro He

 460 465 470 475

 CTC AAC CAG ACC AAG AAC AGA GAC AGC TAC AGC CTG CAT TGG GAA ACT 1536

Leu Asn Gin Thr Lys Asn Arg Asp Ser Tyr Ser Leu His Trp Glu Thr

 480 485 490

 CAG AAA ATA CCC AAA TAC ATT GAT CAC ACT TTC CAG GTC CAG TAC AAG 1584 Gin Lys lie Pro Lys Tyr He Asp His Thr Phe Gin Val Gin Tyr Lys

 495 500 505

 AAG AAG TCA GAG AGC TGG AAG GAC AGC AAG ACA GAA AAC CTA GGT CGA 1632 Lys Lys Ser Glu Ser Trp Lys Asp Ser Lys Thr Glu Asn Leu Gly Arg

-510 515 520

 GTC AAT AGC ATG GAC CTG CCC CAG CTG GAG CCG GAC ACC TCA TAC TGC 1680 Val Asn Ser Met Asp Leu Pro Gin Leu Glu Pro Asp Thr Ser Tyr Cys

 525 530 535

 GCC AGG GTC AGG CTC AAG CCC ATC TCT GAC TAC GAC GGG ATC TGG AGC 1728 Ala Arg Val Arg Val Lys Pro lie Ser Asp Tyr Asp Gly 〖le Trp Ser

 540 545 550 555

 GAG TGG AGC AAT GAC TAC ACT TGG ACG ACT GAC TGG GTG ATG CCC ACG 1776 Glu Trp Ser Asn Glu Tyr Thr Trp Thr Thr Asp Trp Val Met Pro Thr

 560 565 570

 SEQ ID NO: 19

 Sequence length (SEQUENCE LENGTH): 571

 Sequence type (SEQUENCE TYPE): amino acid

 Sequence type (MOLECULE TYPE): Protein

 Sequence (SEQUENCE DESCRIPTION):

 Gin Gly Thr Asn Ser lie Ser Glu Ser Leu Lys Leu Arg Arg Arg Val

 1 5 10 15

His Glu Thr Asp Lys Asn Cys Ser Glu Cly Leu Tyr Gin Gly Gly Pro 20 25 30

 Phe Cys Cys Gin Pro Cys Gin Pro Gly Lys Lys Lys Val Glu Asp Cys

 35 40 45

 Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro Cys Thr Glu Gly Lys

50 55 60

 Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys Cys Arg Arg Cys Thr 65 70 75 80

Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu Thr Asn Cys Thr Leu

 85 90 95

Thr Gin Asn Thr Lys Cys Lys Cys Lys Pro Asp Phe Tyr Cys Asp Ser

 100 105 110

 Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser Cys Glu His Gly Thr

 115 120 125

 Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn Cys Arg Lys Gin Ser

130 135 140

 Pro Arg Asn Arg Leu Trp His Val Thr Glu Glu Glu Glu Thr Val Pro 145 150 155 160

Leu Lys Thr Leu Glu Cys Tyr Asn Asp Tyr Thr Asn Arg lie He Cys

 165 170 175

Ser Trp Ala Asp Thr Glu Asp Ala Gin Gly Leu He Asn Met Thr Leu

 180 185 190

 Leu Tyr His Gin Leu Asp Lys lie Gin Ser Val Ser Cys Glu Leu Ser

 195 200 205

 Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val Pro

210 215 220

 Arg Arg Cys Val lie Pro Tyr Thr Arg Phe Ser Asn Gly Asp Asn Asp 225 230 235 240

Tyr Tyr Ser Phe Gin Pro Asp Arg Asp Leu Gly He Gin Leu Met Val 245 250 255

Pro Leu Ala Gin His Val Gin Pro Pro Pro Pro Lys Asp He His lie

 260 265 270

 Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu Gly

 275 280 285

 Asp Ser Gin Val Ser Trp Leu Ser Ser Lys Asp lie Glu Phe Glu Val

290 295 300

 Ala Tyr Lys Arg Leu Gin Asp Ser Trp Glu Asp Ala Ser Ser Leu His 305 310 315 320

Thr Ser Asn Phe Gin Val Asn Leu Glu Pro Lys Leu Phe Leu Pro Asn

 325 330 335

Ser l ie Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser Ser

 340 345 350

 Leu Ser Gly Arg Pro Ser Arg Trp Ser Pro Glu Val His Trp Asp Ser

 355 360 365

 Gin Pro Gly Asp Lys Ala Gin Pro Gin Asn Leu Gin Cys Phe Phe Asp

370 375 380

 Gly lie Gin Ser Leu His Cys Ser Trp Glu Val Trp Thr Gin Thr Thr 385 390 395 400

Gly Ser Val Ser Phe Gly Leu Phe Tyr Arg Pro Ser Pro Ala Ala Pro

 405 410 415

Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val Tyr

 420 425 430

 Thr Arg Tyr Arg Cys Ser Leu Pro Val Pro Glu Pro Ser Ala His Ser

 435 440 445

 Gin Tyr Thr Val Ser Val Lys His Leu Glu Gin Gly Lys Phe lie Met

450 455 460

Ser Tyr Tyr His He Gin Met Glu Pro Pro lie Leu Asn Gin Thr Lys 465 470 475 480

Asn Arg Asp Ser Tyr Ser Leu His Trp Glu Thr Gin Lys lie Pro Lys

 485 490 495

Tyr 〖le Asp His Thr Phe Gin Val Gin Tyr Lys Lys Lys Ser Glu Ser

 500 505 510

 Trp Lys Asp Ser Lys Thr Glu Asn Leu Gly Arg Val Asn Ser Met Asp

 515 520 525

 Leu Pro Gin Leu Glu Pro Asp Thr Ser Tyr Cys Ala Arg Val Arg Val

530 535 540

 Lys Pro lie Ser Asp Tyr Asp Gly 〖le Trp Ser Glu Trp Ser Asn Glu 545 550 555 560

Tyr Thr Trp Thr Thr Asp Trp Val Met Pro Thr

 565 570

Claims

The scope of the claims  1. Include an immobilized anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody that can specifically bind to a soluble Fas antigen to an insoluble support, and a soluble Fas antigen as a standard substance. A special kit for immunological measurement of ferocious Fas antigen.  2. The kit for immunologically measuring a soluble Fas antigen according to claim 1, further comprising a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen.  3. The soluble Fas antigen as a standard consists of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-13 receptor 3 subunit AIC2A or the constant region of the immunoglobulin heavy chain. 3. The kit for immunoassay of a soluble Fas antigen according to claim 1 or 2, which is a chimeric soluble Fas antigen.  4. The anti-Fas monoclonal antibody bound to the insoluble support is produced from a hybridoma selected from the group consisting of the hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, imported and 〇1111. The kit for immunological measurement of a soluble Fas antigen according to any one of claims 1 to 3, which is a monoclonal antibody.  5. The second anti-Fas monoclonal antibody is a monoclonal antibody produced from a hybridoma selected from the group consisting of the hybridoma VB3, CBE, WB3, ZB4, UB2, AX 6. JAE and CHI1 The immunoassay kit for a soluble Fas antigen according to any one of claims 2 to 4.  6. The labeled second anti-Fas monoclonal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, & D-galactosidase, microperoxidase, alkaline phosphatase and biotin. Item 6. The kit for immunologically measuring a soluble Fas antigen according to any one of Items 2 to 5. 7. Soluble Fa sg 5 characterized by including the following steps a) to c) and d) to; A method for immunological measurement of an antigen. a) A solid-phased anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fs antigen to an insoluble support, and a soluble Fs antigen as a standard substance are added to the immobilized anti-Fas monoclonal antibody. After reacting, b) reacting a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen; c) Measure the amount of labeling of the reaction product Creating a calibration curve by d) After reacting the sample with an immobilized anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fs antigen to an insoluble support,  e) reacting a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fs antigen,  f) Measure the amount of labeling of the reaction product, A step of quantifying a soluble F ss antigen contained in the sample from the calibration curve.  8. The soluble Fa s antigen as a standard substance is the extracellular region of human or mouse Fa s antigen and the extracellular region of mouse IL-13 receptor 1/3 subunit AIC 2A or the constant region of the immunoglobulin heavy chain. 8. The method for immunologically measuring a soluble Fas antigen according to claim 7, which is a chimeric soluble Fas antigen comprising:  9. The anti-Fas monoclonal antibody conjugated to the insoluble support was prepared by the hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CH. A method for immunologically measuring a soluble F s antigen according to claim 7 or 8, which is a monoclonal antibody produced from a hybridoma selected from the group consisting of 11 10. The second anti-Fas monoclonal antibody is a monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CHI1. Item 10. The method for measuring a soluble Fas antigen according to any one of Items 7 to 9. 1 1. The labeled second anti-Fas monoclonal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, β-D-galactosidase, microperoxidase, alkaline phosphatase, and biotin. The method for immunologically measuring a soluble Fas antigen according to any one of claims 7 to 10, which is an antibody.  1 2. Chimeric soluble F s ί 原 原 原 原 原 原 原 原 原 原 原 か ら か ら か ら か ら か ら か ら か ら か ら か ら ヒ ト ヒ ト ヒ ト ヒ ト ヒ ト ヒ ト 2 ..  13. The chimeric soluble Fs antigen according to claim 12 having the amino acid sequence shown in SEQ ID NO: 11 or 19 in the sequence listing.  1 4. A DNA encoding the chimeric soluble Fas antigen according to claim 12 or 13.  15. A DNA encoding the chimeric soluble Fs antigen according to claim 12 or 13, having the DNA sequence shown in SEQ ID NO: 10 or 18. 1 6. An expression vector containing the DNA according to claim 14 or 15.  1 7. A transformed cell transformed with the expression vector according to claim 16.  1 8. Transformed cell which is a cell line identified by International Accession No. FERM BP—4436 or 4437.  1 9. The transformed cell according to claim 18 or 19, wherein the transformed cell is cultured in a medium, and a protein produced in the culture supernatant is obtained. Item 14. The method for producing a chimeric soluble Fas antigen according to item 13 or 13.  20. Obtained by cell fusion of an antibody-producing cell obtained from a mammal immunized with the chimeric soluble Fas antigen according to claim 12 or 13 and a mammalian myeloma cell line. Fusion cells.  2 1. International Deposit No. F ERM BP—A fused cell that is a cell line identified by 4394, 4667 or 4756. 22. A monoclonal antibody reactive to the chimeric soluble Fas antigen according to claim 12 or 13. 23. The monoclonal antibody according to claim 22, which is produced by the fusion cell according to claim 20 or 21.  g 8