CN1271007A - New human chitinase protein and its code sequence - Google Patents

Abstract

The present invention provides a novel human hchi-a protein and its coding sequence expressed in human normal hepatocarcinoma tissue, the present invention also provides the preparation method of the protein and nucleic acid sequence, and the detection of human hchi-a in samples Methods for Nucleic Acid Sequences and Polypeptides.

Description

A Novel Human Chitinase Protein and Its Coding Sequence

The invention relates to the fields of molecular biology, enzymology, physiology, genetic engineering and the like. Specifically, the present invention relates to a hchi-a protein expressed in human liver paracancerous tissue and its nucleic acid sequence. The present invention also relates to the preparation method and use of the protein and nucleic acid sequence.

There are many kinds of chitinases (chitinase, chi) that can bind and degrade insoluble chitin, forming a large family, and its members are found in most organisms. In mammals, it has been reported that chitinase has been detected in atherosclerotic platelets; an increase in the amount of chitinase has also been observed in the blood of Guinea pigs infected with Aspergillus. (J Biol Chem 2000 Jan 7;275(1):514-20). In a variety of insects, chitinase plays an important role in their larval molt growth process, which of course includes Anopheles gambiae, which transmits human malaria, and so on. (J Biol Chem 1997 Nov14; 272(46): 28895-900)

In the present invention, we cloned the homologous gene hchi-a of the chitinase chi-a gene of Aspergillus nidulans in human hepatocarcinoma tissue.

Before the publication of the present invention, the human hchi-a protein sequence and its nucleic acid sequence mentioned in this patent application have not been disclosed or reported.

The first object of the present invention is to provide a new human gene hchi-a (Genbank Accession No.AF212229), which is a human hchi-a protein gene.

The second object of the present invention is to provide a new human protein hchi-a.

The third object of the present invention is to provide a method for producing the above-mentioned novel human hchi-a protein and nucleic acid sequence using recombinant technology.

The present invention also provides the application of the human hchi-a protein polypeptide and coding sequence.

In one aspect of the present invention, a kind of isolated DNA molecule is provided, and this molecule comprises: the nucleotide sequence of the polypeptide that coding has human hchi-a protein activity, described nucleotide sequence and SEQ ID NO.6 There is at least 70% homology to the nucleotide sequence of the DNA molecule from nucleotide 157-1338 in; or the nucleotide sequence can be obtained from the nucleus in SEQ ID NO.6 under moderately stringent conditions The nucleotide sequence of nucleotide 157-1338 is hybridized. Preferably, the sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.7. More preferably, said sequence has the nucleotide sequence from nucleotide 157-1338 in SEQ ID NO.6.

In another aspect of the present invention, an isolated human hchi-a protein polypeptide is provided, which includes: a polypeptide having an amino acid sequence of SEQ ID NO.7, or a conservative variant polypeptide thereof, or an active fragment thereof, or active derivatives. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.7.

In another aspect of the present invention, a vector comprising the above-mentioned DNA molecule is also provided.

In another aspect of the present invention, a host cell transformed with the above vector is also provided. In one example the host cell is E. coli; in another example, the host cell is a eukaryotic cell.

In another aspect of the present invention, there is also provided a method for producing a polypeptide having human hchi-a protein activity, the steps of which are as follows:

(1) The nucleotide sequence encoding the polypeptide having human hchi-a protein activity is operably connected to the expression control sequence to form a human hchi-a protein expression vector, and the nucleotide sequence is the same as SEQ ID NO.6 There is at least 70% homology to the nucleotide sequence from nucleotide 157-1338 in the

(2) transfer the expression vector in the step (1) into the host cell to form a recombinant cell of the human hchi-a protein;

(3) culturing the recombinant cells in step (2) under conditions suitable for expressing the human hchi-a protein polypeptide;

(4) Isolating a polypeptide having human hchi-a protein activity.

Preferably, the nucleic acid sequence used in the method has the sequence of positions 157-1338 in SEQ ID NO.6.

The invention also provides an antibody specifically binding to the hchi-a protein polypeptide.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "human hchi-a protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human hchi-a protein activity, such as the 157th-1338th nucleoside in SEQ ID NO.6 Acid sequences and their degenerate sequences. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 157-1338 nucleotides of the coding frame of the SEQ ID NO.6 sequence . Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% with the 157th-1338th nucleotide sequence in SEQ ID NO.6 can also encode the sequence described in SEQ ID NO.7 . The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 157-1338 in SEQ ID NO.6 under moderately stringent conditions, preferably under highly stringent conditions. The term also includes at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 70% homology with the nucleotide sequence from nucleotide 157-1338 in SEQ ID NO.6 At least 95% nucleotide sequence.

The term also includes variants of the open reading frame sequence of SEQ ID NO. 6 that encode a protein that has the same function as native human hchi-a. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. or wet weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "human hchi-a protein or polypeptide" refers to the polypeptide having the sequence of SEQ ID NO.7 of human hchi-a protein activity. The term also includes variations of the sequence of SEQ ID NO. 7 that have the same function as natural human hchi-a. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of human hchi-a protein.

Variants of the human hchi-a polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and human hchi-a DNA under high or low stringent conditions The protein encoded by the hybridized DNA, and the polypeptide or protein obtained by using the antiserum against human hchi-a polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human hchi-a polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human hchi-a polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human hchi-a polypeptide sequence. At least about 100 contiguous amino acids.

In the present invention, "human hchi-a conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO.7, at most 10, preferably at most 8, more preferably at most 5 amino acids are similar in nature or similar amino acids to form polypeptides. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The invention also includes analogs of human hchi-a protein or polypeptide. The difference between these analogues and the natural human hchi-a polypeptide may be the difference in amino acid sequence, or the difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the human hchi-a polypeptide of the present invention, the human hchi-a coding sequence can be operably linked to the expression control sequence, thereby forming a human hchi-a protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.

The invention also provides antibodies specifically binding to human hchi-a, including polyclonal antibodies and monoclonal antibodies.

In the present invention, a series of methods known in the art can be used to prepare antibodies specific to human hchi-a. For example, the purified human hchi-a gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human hchi-a or its antigenic fragments can also be used to immunize animals to produce antibodies. Antibodies produced according to the present invention may also be monoclonal antibodies, which can be produced using hybridoma technology (e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6 : 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976). The antibody of the present invention includes an antibody that can suppress the function of human hchi-a, and can also be an antibody that does not affect the function of human hchi-a. Each type of antibody can be produced by immunizing the fragment or functional domain of the human hchi-a gene product, and the human hchi-a gene product and its fragment can be produced by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind unmodified forms of the human hchi-a gene product can be obtained by immunizing animals with the gene product produced in prokaryotic cells such as E. coli. Antibodies that bind to post-translationally modified forms, such as glycosylated or phosphorylated proteins or polypeptides, can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.

The human hchi-a antibody of the present invention can be used to identify cells expressing human hchi-a protein or polypeptide, such as Jurkat T cells. For example, human hchi-a-specific antibodies can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and then human hchi-a-specific antibodies can be contacted with a cell sample, and then analyzed by fluorescence microscopy or flow cytometry. The instrument detects the cells combined with human hchi-a specific antibody.

In addition to detection of human hchi-a on the cell surface, the protein can also be analyzed by Western blotting. Cell lysates can be extracted from cultured cells or tissue samples taken from patients such as liver paracancerous tissue, and dissolved in lysis buffer containing detergent. Cell extracts were then separated by SDS polyacrylamide gel electrophoresis (with purified human hchi-a polypeptide as a positive control), followed by transfer to nitrocellulose by electrophoretic hybridization. For immunodetection of human hchi-a polypeptide by Western blot, typical antibody binding detection methods such as autoradiography or alkaline phosphatase detection methods can be used. Inoculation serum or irrelevant monoclonal antibodies can be used as controls for non-specific reactions.

The expression of human hchi-a gene product can also be analyzed by Northern blot technique, that is, the presence or absence and quantity of human hchi-a RNA transcripts in cells can be analyzed.

Northern blot analysis of human hchi-a DNA and Western blot analysis of human hchi-a specific antibody can be used in combination to confirm the expression of human hchi-a in biological samples. Human hchi-a DNA can also be used for Southern blot analysis or in situ hybridization analysis to locate the gene on the chromosome, and genetic linkage analysis can be performed to find out other possible disease-related genes.

In addition, the present invention also provides a nucleic acid molecule that can be used as a probe. The molecule usually has 8-100 consecutive nucleotides of the human hchi-a nucleotide coding sequence, preferably 15-50 consecutive nucleotides. Nucleotides. The probe can be used to detect whether there is a nucleic acid molecule encoding human hchi-a in a sample.

The present invention also provides a method for detecting whether there is human hchi-a nucleotide sequence in a sample, which comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the human hchi-a nucleotide coding sequence and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the human hchi-a nucleotide sequence and amino acid sequence of the present invention, human hchi-a homologous genes or homologous proteins can be screened on the basis of nucleic acid homology or expressed protein homology.

In order to obtain a dot array of human cDNAs or genomic DNAs related to the human hchi-a gene, the human cDNA or genomic DNA library can be screened with DNA probes that are tested against human hchi-a with ³² P under low stringency conditions. All or part of it is radioactively labeled. The most suitable cDNA library for screening is the library from human liver paracancerous tissue. cDNA libraries from other human tissues involved in endocrine or specific human cell lines may also be used for screening purposes. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, for example, from Clontech, Palo Alto, Cal. This screening method can identify the nucleotide sequences of gene families related to human hchi-a.

Screening for human hchi-a homologues based on nucleotide similarity can be accomplished as follows. Human liver paracancerous tissue cDNA library, such as Clontech Cat.#74XX (Clontech, Palo Alto, Cal.) can be screened with a random primer DNA probe containing all or part of the human hchi-a gene sequence. To complete the identification of clones with DNA insertion sequences at least 70% homologous to the human hchi-a sequence, a hybridization solution with a hybridization temperature of 55° C. can be used, followed by washing with 0.5×SSC and 0.1% SDS. The cloned DNA insert identified in this way can be further evaluated for its similarity to the human hchi-a gene by DNA restriction enzyme analysis and DNA sequencing. The distribution of tissue expression can be analyzed using the Northern blotting technique described above.

Human hchi-a homologs can also be identified with antibodies against human hchi-a protein or polypeptide. For example, commercially available or constructed expression libraries derived from cells or tissues such as hepatic paracancerous tissues can be screened by standard methods. The library is poured into a plate, and the colony is transferred to a nitrocellulose membrane, allowing the expressed recombinant protein to bind to the membrane. Typical antibody binding and detection can then be performed with specific human Hchi-A antibodies. The DNA insert in the clone identified by this method can be further analyzed by DNA restriction enzyme analysis and DNA sequencing to evaluate its similarity to the human hchi-a gene. Tissue expression profiles of newly identified genes can be analyzed similarly as described above.

The full-length human hchi-a nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial chemical synthesis. Before this application, in the prior art, it was possible to obtain the nucleic acid sequence encoding the human hchi-a protein of the present invention by first synthesizing multiple small polynucleotide fragments and then connecting them. Then, the nucleic acid sequence can be introduced into various existing DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct peptide synthesis using solid-phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

The coding sequence of the protein of the present invention can be used for gene mapping. For example, by fluorescent in situ hybridization (FISH), cDNA clones can be hybridized with metaphase chromosomes to accurately locate chromosomes. The technique can use cDNAs as short as about 500 bp; cDNAs as long as about 2000 bp or longer can also be used. For this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise location on a chromosome, it will be possible to correlate the sequence's physical location on the chromosome with genetic map data. Such genetic map data are available, for example, through the Mendelian Human Genetics Database (available online through the Johns Hopkins University Welch Medical Library). Linkage analysis is then used to identify associations between genes and diseases that have been mapped to the same chromosomal region.

Next, it is necessary to determine the differences in cDNA or genome sequence between diseased and healthy individuals. If a mutation is present in some or all affected individuals but not in normal individuals, the mutation may be the cause of the disease.

Using the human hchi-a protein of the present invention, through various conventional screening methods, substances that interact with human hchi-a, or receptors, inhibitors or antagonists can be screened out.

When the human hchi-a protein of the present invention and its antibody, inhibitor, antagonist, or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be adjusted according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the human hchi-a protein of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of protein and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The human hchi-a protein of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When the human hchi-a protein polypeptide of the present invention is used as a medicine, a therapeutically effective dose of the polypeptide can be administered to mammals, wherein the therapeutically effective dose is usually at least about 10 micrograms per kilogram of body weight, and in most cases no More than about 8 mg/kg body weight, preferably the dose is about 10 microgram/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Table 2 is a homology comparison (GAP) table of the nucleotide sequences (GenBank Accession No.D87063) of the human hchi-a protein of the present invention and the Aspergillus nidulanschi-a protein.

Table 3 is a homology comparison (FASTA) table of the amino acid sequences (GenPept Accession No.BAA35140) of the human hchi-a protein of the present invention and the Aspergillus nidulanschi-a protein. Among them, the same amino acid is marked with a single amino acid character between the two sequences, and the similar amino acid is marked with "+".

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, generally according to routine conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

Cloning of human hchi-a gene

1. Tissue separation (paracancer tissue isolation)

The paracancerous tissues were obtained from 5 normal adult male donors. The paracancerous tissues were taken out within four hours after death and immediately placed in liquid nitrogen for cryopreservation.

2. Isolation of mRNA (mRNA isolation)

Take out the tissue, grind it with a mortar, add it into a 50ml tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a new 50ml tube and extract total RNA (TRIzol Reagents, Gibco, NY, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis. The mRNA in the total RNA was separated and quantified using a cellulose column with Oligo d(T).

3. Construction of cDNA library

Using mRNA as a template, double-stranded cDNA is synthesized, and the reverse transcription primer is shown in SEQ ID NO.1. After blunting the ends, add adapters containing EcoRI cutting points, and the adapter sequences are shown in SEQ ID NO.2 and 3, respectively. After phosphorylation of the EcoRI terminus, it was digested with XhoI restriction endonuclease for 1.5 hours before fragment isolation. Fragments with a length >500bp were screened through the column, extracted with phenol-chloroform, precipitated with ethanol, dissolved in sterile water, connected to the Uni-ZAP XR vector (Stratagene, CA9203, USA), and Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene , CA9203, USA) were packaged, and the host bacteria used XL 1-Blue MRF (Stratagene, CA9203, USA) bacteria. Plate and measure titer.

4. Sequencing and Database Constructing

The clones with foreign fragments inserted in the library were selected, and the plasmids were extracted after amplification (Qiagen, Germany). T3 and T7 were used as universal primers at the 3' and 5' ends, and terminators were fluorescently labeled (Big-Dye, Perkin- Elmer, USA) method, EST large-scale sequencing was carried out on ABI 377 sequencer (Perkin-Elmer, USA). The sequencing results were removed from the carrier sequence by FACTURA software and transferred to SUN Ultra 450 Server for further processing. All the sequence information is then searched in the existing database (Genebank+EMBL) with BLAST and FASTA software in the GCG software package (Wisconsin group, USA), and sequences with no homology or homology lower than 95% are regarded as new Gene database.

5. Cloning of Full-length cDNA

On the basis of the obtained new gene fragment sequence information, the cDNA full-length clone is carried out in two stages:

(1) "Electronic Cloning"

Use the new gene fragment sequence as a probe to search the dbEST database, and consider the expressed sequence tag (Expressed Sequence Tag, “EST” for short) sequence with an overlapping sequence > 50bp and a homology of more than 98% as the same sequence (consensus sequence), take it out and use AUTOASSEMBLER software for splicing, part of EST can extend the probe sequence. Then use STRIDER software to analyze whether the extended sequence has a complete open reading frame (Open Reading Frame, ORF), and use BLAST to search Genbank or SwissProt to determine whether the sequence is homologous to other species at the nucleotide and amino acid levels , to help determine the full-length integrity of the obtained gene. By means of electronic cloning, the full-length sequence of the human hchi-a gene can usually be obtained.

(2) Rapid amplification of cDNA ends (Rapid Amplification of cDNA Ends, RACE)

If the complete cDNA full-length has not been obtained by the "electronic cloning" method, then design primers at the 5' or 3' end of the existing sequence, and use it in the Marathon-Ready cDNA library (Clontech Lab, Inc, USA) of human liver paracancerous tissue. Perform long-range PCR reactions. The PCR products were then cloned and sequenced. Use AUTOASSEMBLER and STRIDER software to analyze whether the extended sequence has a complete ORF, if not, repeat the above process until the full length is obtained.

(3) RT-PCR

For the known sequences at the 5' and 3' ends, if there is still a gap in the middle that cannot be obtained from the existing public database or its own database, RT-PCR may be considered. Primers were designed at the 5' end of the sequence, and Oligo-dT was used as a primer at the 3' end to amplify in the total RNA pool of adjacent liver cancer tissues. The products were then cloned and sequenced. Finally splice and get the full length.

By using the above three methods in combination, the full-length coding sequence of the candidate human hchi-a protein was obtained. On the basis of splicing to obtain the full length (including at least the complete open reading frame), further design primer R1: 5'-GACATGCAGATTTCCACCCAG-3' (SEQ ID NO.4) as the forward primer, oligonucleotide R2: 5' -TTCACACCTGCTCACTCACTCC-3' (SEQ ID NO.5) was used as a reverse primer, and the total RNA of the paracancerous tissue was used as a template for RT-PCR amplification. The PCR conditions of R1/R2 were 94°C for 5 minutes, followed by 94 35 cycles of 30 seconds at 59°C, 30 seconds at 59°C and 1 minute at 72°C, with a final extension at 72°C for 5 minutes. The PCR amplification product was detected by electrophoresis, and the length of the amplified fragment was 1406bp. Then, the PCR amplification product was cloned and sequenced according to conventional methods to obtain the sequence shown in SEQ ID NO.6.

Example 2

Sequence information and homology analysis of human hchi-a gene:

The novel human hchi-a full-length cDNA of the present invention (GenBank Accession No.AF212229. It has not been disclosed before this application) has a length of 1406bp, and the detailed sequence is shown in SEQ ID NO.6, wherein the open reading frame is located at the 157-1338 core glycosides. The amino acid sequence of human hchi-a was deduced according to the full-length cDNA, with a total of 393 amino acid residues, a molecular weight of 44968.68, and a pI of 8.74. See SEQ ID NO.7 for the detailed sequence.

The full-length cDNA sequence of hchi-a and its encoded protein were carried out by BLAST program in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases for nucleotide and protein synchronization According to the source search, it was found that it has a certain homology with the chi-a gene of Aspergillus nidulans. At the nucleotide level, it has 51.4% identity (attached table 2) to the mRNA full coding sequence of the Aspergillus nidulanschi-a gene (GenBank Accession No.D87063), and at the amino acid level, it is identical to the Aspergillus nidulanschi-a protein (GePept Accession No.BAA35140) amino acid residues 105-373 have 23.6% identity and 56.9% similarity (Supplementary Table 3). It can be seen from the above that the hchi-a gene and the Aspergillus nidulanschi-a gene have high homology in both nucleic acid and protein levels, and it can be considered that the two are also highly similar in function.

The human hchi-a of the present invention can be used as a member of the family for further functional research, and can also be used to produce fusion proteins with other proteins, such as immunoglobulins to produce fusion proteins. In addition, the human hchi-a of the present invention can also be fused or exchanged fragments with other members of the family to produce new proteins. For example, the N-terminal of the human hchi-a protein of the present invention is exchanged with the N-terminal of the Aspergillus nidulanschi-a protein to produce new proteins with higher activity or new properties.

The antibody against human hchi-a of the present invention is used for screening other members of this family, or for affinity purification of related proteins (such as other members of this family).

In addition, the human hchi-a nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human hchi-a or inhibit the overexpression of human hchi-a. The human hchi-a protein or its active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by the lack, non-function or abnormality of human hchi-a. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for relevant diagnosis or prognosis.

Since the human hchi-a protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or when administered to humans, compared to homologous proteins derived from other species (such as Aspergillus nidulans) or lower side effects (eg, lower or no immunogenicity in humans).

Example 3

Tissue expression profile of human hchi-a gene

Electronic Northern Expression Profiles. According to Ton C. et al.'s method (Ton C et al., Biochem BiophysRes Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997 Dec 16; 96 (12): 4146-4203 ), the human hchi-a cDNA sequence was searched by BLAST in the humanEST database in the GCG software package. Among the obtained human ESTs, there were 81 ESTs with probability values <10e-10 and identity >95%, which can be regarded as The transcriptional expression of the gene in the tissue, from which the tissue spectrum of the gene expression can be obtained, and it is found that it is expressed in tissues such as lung cancer, pineal gland, spleen, placenta, infant heart, fetal liver, testis, fetal brain, etc. , indicating that it plays an important role in these tissues and organs of the human body.

Example 4

Preparation and purification of human hchi-a polypeptide

In this example, the full-length human hchi-a coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

Prokaryotic expression of human hchi-a polypeptide in the form of GST fusion protein in Escherichia coli.

Construction of prokaryotic expression vector and transformation of Escherichia coli

According to the full-length coding sequence (SEQ ID NO.6) of human hchi-a, design primers that amplify the complete coding reading frame (corresponding to about 20 or more nucleotides at the 5' and 3' ends of the coding sequence, respectively), and Restriction endonuclease sites (this depends on the selected pGEX-2T vector) were introduced on the forward and reverse primers respectively, so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the human hchi-a gene was cloned into the pGEX-2T vector (Pharmacia, Piscataway, NJ) under the premise of ensuring the correct reading frame. The identified expression vector was transformed into Escherichia coli DH5α using the CaCl ₂ method, and the engineering strain DH5α-pGEX-2T-hchi-a containing the pGEX-2T-hchi-a expression vector was screened and identified.

Isolation and Identification of Engineering Bacteria Expressing GST-hchi-a Recombinant Protein

Pick the DH5α-pGEX-2T-hchi-a engineered bacterium of single bacterium in 3ml containing the LB medium of 100 μ g/ml ampicillin and shake and cultivate overnight, draw the culture solution in the new LB medium ( containing 100 μg/ml ampicillin) for about 3 hours, until the OD ₆₀₀ reaches 0.5, add IPTG to a final concentration of 1 mmol/L and continue to culture at 37°C for 0, 1, 2, 3 hours respectively. Centrifuge 1ml of bacterial solution with different culture time, add lysate (50μl of 2×SDS loading buffer, 45μl of distilled water, 5μl of dimercaptoethanol) to the bacterial sediment, suspend the bacterial sediment, boil in boiling water bath for 5 minutes, 10000rpm Centrifuge for 1 minute, add the supernatant to 12% SDS-PAGE gel electrophoresis. After staining, it is observed that the amount of protein with expected molecular weight increases with the increase of IPTG induction time, which is the engineering bacteria expressing GST-hchi-a fusion protein.

Extraction and purification of GST-hchi-a fusion protein

The engineered bacteria DH5α-pGEX-2T-hchi-a expressing GST-hchi-a fusion expression protein was induced by the above method. The induced bacteria were centrifuged and precipitated, and 20ml of PBS was added to resuspend the bacteria for every 400ml of bacteria, and the bacteria were sonicated. Add 50% glutathione Sepharose 4B saturated with PBS in an amount of 20 microliters per milliliter to the completely broken bacteria, shake at 37°C for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes to precipitate glutathione bound to GST-hchi-a Glycoside peptide Sepharose 4B, discard the supernatant. Add 100 μl of PBS to the precipitate obtained in each milliliter of ultrasonic liquid to wash twice, then add 10 μl of reduced glutathione eluent to the precipitate obtained in each milliliter of ultrasonic liquid, place at room temperature for 10 minutes, centrifuge at 10,000 rpm for 10 minutes, and the supernatant is Eluted fusion protein. Repeat the elution twice. The eluted supernatant was stored at -80°C, and subjected to SDS-PAGE electrophoresis to detect the purification effect. The protein band at 45kDa is human hchi-a protein.

Example 5

Eukaryotic expression of human hchi-a protein or polypeptide in insect cells

1. Construction of human hchi-a baculovirus expression vector and transfection of Sf9 insect cell line

According to the full-length coding sequence (SEQ ID NO.6) of human hchi-a, design the primer that amplifies the complete coding reading frame, and introduce restriction endonuclease sites respectively on forward and reverse primers (this can be selected vectors) in order to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hchi-acDNA was cloned into pVL1392 vector (Invitrogen, Carlsbad, CA) under the premise of ensuring the reading frame. Add 3 μg of the identified expression vector, 1 μg of wild-type linear baculovirus DNA (BaculoGold ^TM ACMNPV DNA, Pharmingen, San Diego, CA) and 25 μl of Lipofection (Gibco-BRL, NY) to 1 ml of serum-free insect medium, shake Mix for 15 seconds and incubate at room temperature for 15 minutes. Take 1ml (2×10 ⁶ ) of Sf9 insect cell suspension in a 60mm tissue culture plate, change the transfection medium after 1 hour of attachment, discard the medium after incubating at room temperature for 15 minutes, and add the prepared DNA vector transfection mixture , Seal the culture plate with Parafilm, shake and culture at room temperature 27°C for 4 hours, then change the complete medium and culture for 3 days, and collect the supernatant for use.

2. Screening and identification of insect cell lines transformed into recombinant expression vectors

Insect cells 3 days after transfection were made into cell suspension (2×10 ⁶ /1ml) with fresh medium, 1ml of cell suspension was placed in a 60mm tissue culture dish, 3ml of medium was added, and 100μl of culture supernatant collected, Adhere to the wall for 1 hour, discard 2ml of medium, continue to culture at room temperature for 1 hour, discard all the medium, add 3ml of preheated semi-solid medium containing 20μl of 4% X-gal, pick white cell clones after 5-7 days of culture Cultured in a 96-well culture plate for 3-5 days, and then aspirate the supernatant to infect Sf9 insect cells.

Infected cells were collected for Western identification. After the cells were lysed, SDS-PAGE electrophoresis was performed. The gel after electrophoresis was transferred to the nitrocellulose membrane in Pharmacia's Multiphor II semi-dry electrotransfer instrument, and the nitrocellulose membrane was placed in the blocking solution for blocking for 1 hour, and then in Block in the anti-human hchi-a antibody solution for 1 hour, shake and wash with TBS solution for 5 minutes, a total of 2 times, and then place the membrane in the biotin-labeled anti-human hchi-a primary antibody secondary antibody solution and shake for 1 hour , wash with TBS, add avidin-alkaline phosphatase complex to react for 30 minutes, wash twice with TBS, add freshly prepared chromogenic solution to develop color and observe protein bands.

Sf9 cell clones that highly express human hchi-a were picked.

3. Extraction and purification of human hchi-a protein

Sf9 cells were massively infected with the supernatant of Sf9 cell clones highly expressing human hchi-a, and the cells were collected 48 hours after infection and washed with PBS. Add 20ml of cell lysate (0.5% Triton X-100, 20mM Na ₃ PO ₄ (sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃ VO ₄ (sodium vanadate), 1mM Pefabloc, for every 2×10 ⁸ cells 1 μg/ml pepsin, leupeptin and aprotinin) to break the cells, centrifuge at 12000×g for 20min to remove cell debris, add 2ml NTA-agarose (Qiagen, Germany) to the supernatant for every 2× ¹⁰⁸ cells , adsorption at 4°C for 1 hour. Then, it was washed twice with His buffer containing 100 nM imidazole, and eluted with a buffer containing 20 mM N,N'-dipiperazine, 500 mM NaCl, and 300 mM imidazole to obtain a purified protein. The eluate was stored at 4°C, and the purity of the extracted human hchi-a protein was detected by SDS-PAG electrophoresis. The protein band at 45kDa is human hchi-a protein.

Example 6

Preparation of anti-human hchi-a antibody

1. Preparation of immunized mice and splenocytes: the human hchi-a protein obtained in Example 5 and Example 6 is separated by chromatography for subsequent use, and can also be separated by SDS-PAGE gel electrophoresis. Electrophoretic bands were excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Take 6-8 week-old Balb/C female mice, and inject them intraperitoneally with 50-100 μg/0.2ml emulsified protein. After 14 days, the mice were boosted with the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 μg/0.2ml, and used for fusion after 3-5 days. Among them, for the preparation of spleen cells, see E Zheng, editor-in-chief, "Tissue Culture and Molecular Cytology", Beijing Publishing House, p. 210.

2. Prepare feeder cells according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 371.

3. Carry out cell fusion according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 213.

4. Antibody detection: After 10-15 days of cell fusion, it is necessary to check well by well. Once vigorous hybrid cell colony growth is found, human hchi-a protein is used for preliminary screening of antibody activity. The commonly used methods are: immunofluorescence test, emission immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). Immediately after the wells with antibody activity were detected, the clones were cultured and the antibodies were isolated.

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequential features:

(A) Length: 50bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.1GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

(2) Information of SEQ ID NO.2

(i) Sequential features:

(A) Length: 13bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.2

AATTCGGCAC GA G 13

(3) Information of SEQ ID NO.3

(i) Sequential features:

(A) Length: 9bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.3

GCCGTGCTC 9(4) Information (i) of SEQ ID NO.4. Sequence characteristics:

(A) Length: 21bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (iii). Sequence description: SEQ ID NO.4GACATGCAGATTTCCACCCAG 21(5) Information of SEQ ID NO.5 (i). Sequence characteristics:

(A) Length: 22bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (iii). Sequence description: SEQ ID NO.5TTCACACCTGCTCACTCACTCC 22 (6) Information of SEQ ID NO.6 (i) Sequence features:

(A) Length: 1406bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: Nucleotide (iii) Sequence description: SEQ ID NO.6

1 GACATGCAGA TTTCCACCCA GAAGACAGAG AAGGAGCCAG TGGTCATGGA

51 ATGGGCTGGG GTCAAAGACT GGGTGCCTGG GAGCTGAGGC AGCCACCGTT

101 TCAGCCTGGC CAGCCCTCTG GACCCCGAGG TTGGACCCTA CTGTGACACA

151 CCTACCATGC GGACACTCTT CAACCTCCTC TGGCTTGCCC TGGCCTGCAG

201 CCCTGTTCAC ACTACCCTGT CAAAGTCAGA TGCCAAAAAA GCCGCCTCAA

251 AGACGCTGCT GGAGAAGAGT CAGTTTTTCAG ATAAGCCGGT GCAAGACCGG

301 GGTTTGGTGG TGACGGACCT CAAAGCTGAG AGTGTGGTTC TTGAGCATCG

351 CAGCTACTGC TCGGCAAAGG CCCGGGACAG ACACTTTGCT GGGGATGTAC

401 TGGGCTATGT CACTCCATGG AACAGCCATG GCTACGATGT CACCAAGGTC

451 TTTGGGAGCA AGTTCACACA GATCTCACCC GTCTGGCTGC AGCTGAAGAG

501 ACGTGGCCGT GAGATGTTTG AGGTCACGGG CCTCCACGAC GTGGACCAAG

551 GGTGGATGCG AGCTGTCAGG AAGCATGCCA AGGGCCTGCA CATAGTGCCT

601 CGGCTCCTGT TTGAGGACTG GACTTACGAT GATTTCCGGA ACGTCTTAGA

651 CAGTGAGGAT GAGATAGAGG AGCTGAGCAA GACCGTGGTC CAGGTGGCAA

701 AGAACCAGCA TTTCGATGGC TTCGTGGTGG AGGTCTGGAA CCAGCTGCTA

751 AGCCAGAAGC GCGTGGGCCT CATCCACATG CTCACCCACT TGGCCGAGGC

801 TCTGCACCAG GCCCGGCTGC TGGCCCTCCT GGTCATCCCG CCTGCCATCA

851 CCCCCGGGAC CGACCAGCTG GGCATGTTCA CGCACAAGGA GTTTGAGCAG

901 CTGGCCCCCG TGCTGGATGG TTTCAGCCTC ATGACCTACG ACTACTCTAC

951  AGCGCATCAG CCTGGCCCTA ATGCACCCCT GTCCTGGGTT CGAGCCTGCG1001  TCCAGGTCCT GGACCCGAAG TCCAAGTGGC GAAGCAAAAT CCTCCTGGGG1051  CTCAACTTCT ATGGTATGGA CTACGCGACC TCCAAGGATG CCCGTGAGCC1101  TGTTGTCGGG GCCAGGTACA TCCAGACACT GAAGGACCAC AGGCCCCGGA1151  TGGTGTGGGA CAGCCAGGTC TCAGAGCACT TCTTCGAGTA CAAGAAGAGC1201  CGCAGTGGGA GGCACGTCGT CTTCTACCCA ACCCTGAAGT CCCTGCAGGT1251  GCGGCTGGAG CTGGCCCGGG AGCTGGGCGT TGGGGTCTCT ATCTGGGAGC1301  TGGGCCAGGG CCTGGACTAC TTCTACGACC TGCTCTAGGT GGGCATTGCG1351  GCCTCCGCGG TGGACGTGTT CTTTTCTAAG CCATGGAGTG AGTGAGCAGG1401  TGTGAA(7) Information of SEQ ID NO.7 (i) Sequence characteristics: (A) Length: 393 amino acids (B) Type: amino acid (C) Chain: single chain (D) Topological structure: linear (ii) Molecular type: polypeptide (iii) )序列描述：SEQ ID NO.7：  1  MRTLFNLLWL ALACSPVHTT LSKSDAKKAA SKTLLEKSQF SDKPVQDRGL51  VVTDLKAESV VLEHRSYCSA KARDRHFAGD VLGYVTPWNS HGYDVTKVFG101  SKFTQISPVW LQLKRRGREM FEVTGLHDVD QGWMRAVRKH AKGLHIVPRL151  LFEDWTYDDF RNVLDSEDEI EELSKTVVQV AKNQHFDGFV VEVWNQLLSQ201  KRVGLIHMLT HLAEALHQAR LLALLVIPPA ITPGTDQLGM FTHKEFEQLA251  PVLDGFSLMT YDYSTAHQPG PNAPLSWVRA CVQVLDPKSK WRSKILLGLN301  FYGMDYATSK DAREPVVGAR YIQTLKDHRP RMVWDSQVSE HFFEYKKSRS351  GRHVVFYPTL KSLQVRLELA RELGVGVSIW ELGQGLDYFY DLL

Table 2Percent Identity: 51.4%

. . . . .1 ............................................ .....G1801 CGCAACTACAACCCCCAAGATCTCCCCGCGGAGAAATTGACCCATATTCT 850

. . . . . . .2 ACATGCAGATTTCCACCCAGAAGACAGAGAAGGAGCCAGTG...GTCATG 48

| ||| | ||| | | |||||| | |||| | | |851 TTACGCA.TTCGCCAATGTCAGGCCAGAGACTGGCGAAGTGTAAGCCGAA 899

. . . . . . .49 GAATGGGCTGGGGTCAAAGACTGGGTGCCTGGGAGCTGAGGCAGCCACCG 98

|||| || || ||| | | | |||| | || | ||| |900 CCATGGACT.ATGTGGAAGCCAGCCCAAGCCGAAGCTAATGCCGACACAG 948

. . . . . . .99 .TTTCAGCCTGGCCAGCCCTCTG.GACCCCGAGGTTGGACCCTACTG... 143

| | | ||| | |||| | | || | |||| ||||949 CTACCTTTCTGACACCTGGTCTGATATCGAGAAGCACTACCCCACTGATT 998

. . . . . . .144 .......TGACACACCTACCA.....TGCGGACAC.TCTTCAACCTCCTC 180

||| || | || | | ||| | || | || |999 CTTGGAATGATACCGGAAACAACGTCTACGGCTGCGTCAAGCAGCTGGGC 1048

. . . . . . .181 TGGCTTGCCCTGGCCTGCAGCCCTGTTCACACTACCCTGTC.AAAGTCAG 229

| |||| | | | ||| | | | | | |||| ||

. . . . . . .230 ATG..CCAAAAAAAGCCGCCT....CAAAGACGCTGCT....GGAGAA 266

||| || | || || ||| | |||| | || |1099 ATGGACCTATTTCTCCCAACTTTACCAACGGAGCTGGTACCCCCTGAGAACA 1148

. . . . . . .267 GAGTCAGTTTT...CAGA.......TAAGCCGGTGCAAGACC.GGGGTTT 305

|||| ||||||||||||||||||||||||1149 GAGCCCGCTTTGCGCAGACTGCGACAAAGCTCATCACTGACCTTGGTTTT 1198

. . . . . . .306 GGTGGTGACGGACCTCAAAGCT.GAGAGTGTGGTTCTTGA.GCATCGCAG 353

| ||||| | |||| || | || || | | | | | || ||

. . . . . . .354 CTACTGCTCGGCAAAGGCCCGGGACAGACACTTTGCTGGGGATGTACT.G 402

||| || |||||||||||||||||||||||1249 CTAATG......GAAGCTCAGGGATCGATA.TTGATTGGGAGTATCCTCA 1291

. . . . . . .403 GGCTATGTCAC...TCCATGGAACAGCCATGGCTAC....GATGTCACCA 445

| || || | | | | | | | ||||

. . . . . . .446 AGGTCTTTGGGAGCAAGTTCACACAGATCTC..ACCCGTCTGGC... 487

|||||||||||||||||||||||1342 CGATCAGCAGGCTCAGAACTACGTGGATCTCCTACGCAGGTGCCGTGAAG 1391

. . . . . . .488 ....TGCAGCTGAAGAGACGTGGCCGTGAGATG...TTTGAGGTCACGGG 530

||||||||||||||||||||||||||1392 TAAGTGCATCACTAGTGACTCGGTCCTTCATTGTGCTAATATGGCTAGGC 1441

. . . . . . .531 CCTCCACGACG...TGGACCAAGGGTGGATGCGAGCTGTCAGGAAGCATG 577

|| |||||||||||||||||||||||1442 TCTGAATGCCGCTCAGGGCCAACGGAGG.TTCCAGCT.TACTGTTGCTGT 1489

. . . . . . .578 CCAAGGGCCTGCACATAGTGCCTCGGCTCCTGTTTGAGGACTGGACTTAC 627

|| || | | | | | ||| | | | || | || | |1490 CCCAGCGGGTAAGTGTGTACCATCGAAGGATCTATGCAGGCT..AACCAG 1537

. . . . . . .628 GATGATTTCCGGAACGTCTTAGACAGTGAGGATGAGATAGAGGAGCTGAG 677

|||||||| ||||||||||||||||||1538 TGCCAGGTCCGGATAAT....TACAATAAGCTCCGGCTACAGGAGATGAC 1583

. . . . . . .678 CAAGACCGTGGTC.........CAGGTGGCAAAGAACCA.GCATTTCGAT 717

| | | | | | | |||| | || | ||

. . . . . . .718 GGCTTCGTGGTGGAGGTCTGGA.ACCAGCTGCTAAGCCAGAAGCGCGTGG 766

|| || | | |||| | | | | | || ||

. . . . . . .767 GCCTCATCCACATGCTCACCCACTTGGCCGAGGCTCTGCACCA....... 809

|||||||||||||||||||||||||||1684 CCCACGTCCACTCCCCTTTAACACT..GTCCAAGCTGTGAACCACTATATC 1731

. . . . . . .810 GGCCCGGCTGCTGGCCC.....TCCTGGTCATCC..CGCCTGCCA..TCA 850

| | | || || ||| | | | |||| || |||||

. . . . . . .851 CCCCCGGGACCGACCAGCTGGGCATGTTCACGCACAAGGAGTTTGAGCAG 900

| ||| | | | | | | || | || || | | |1782 CGGCCGTGCCTTTCAGAACACCGATGGCCCCGGCC...GACCTTACTCTG 1828

. . . . . . .901 CTGGCCCCCGTGCTGGATGGTTTCAGCCTCATGACCTACGACTACTCTAC 950

| | | | ||| ||| | | | || || ||| |1829 GTATAGGCCAAGGGACGTGGGAGCAGGGTGTT....TATGATTAC...AA 1871

. . . . . . .951 AGCGCATCAGCCTGGCCCTAATGCACCCCTGTCCTGGGTTCGAGC....C 996

||||||||||||||||||||||||1872 GGCGCTGC..CCAGACCGGGTGCCACCGAGCAGCTGGATACCAACATTGG 1919

. . . . . . .997 TGCGTCCAGGTCCT.GGACCCGAAGTCC....AAGTGG...CGAAGCAAA 1038

||||||||||||||||||||||||||||||||1920 TGCGTCCTGGTCGTATGATCCTTCGTCCCGTGAAATGGTTTCGTACGATA 1969

. . . . . . .1039 ATCCTCCTGGGGCTCAACTTCTATGGTATGGACTACGCGACCTCCAAGGA 1088

| | | ||| | || || | | | || ||

. . . . . . .1089 TGCCCGTGAGCCTGTTGTCGGGGCCAGGTACATCCAGACACTGAAGGACC 1138

|| || ||| || || | || | | | | | | |||2018 TCGGCGGAGCTATGTGGTGGGAAACTAGTGCTGATAAGGGCGGCAAGACT 2067

. . . . . . . .1139 AC.....AGGC..CCCGGATGGTGTGGGACAGCCAGGTCTCAGAGCACTT 1181

||||||||||||||||||||||2068 GCGAACAAGGCTGACGGCAGCTTGATTGGCACCTTTGTCGAAGATGTTGG 2117

. . . . . . .1182 CTTCGAGTACAAGAAGAGCCGCAGTGGGAGGCACGTCGTCTTTCTACCAA 1231

||||||||||||||||||||||||||2118 AGGCGTCAATAACTTGGATCGCACACAGA...ACGCCATCAGCTACCCCG 2164

. . . . . . .1232 CCCTGAAGTCC.....CTGCAGGTGCGGCTGGAGCTGGCCCGGGAGCTGG 1276

| | | | | | | | | |||| | | | || | | | |2165 ACAGCCAGTACGACAACCTCAAGGCTGGCTTCCCGTCGTCCTAGGGTAAG 2214

. . . . . . .1277 G...CGTTGGGGTCTCTATCTG.GGAGCTG.............GGCCAG 1308

| | | | | || ||| || | ||

. . . . . . .1309 GGCCTGGACTACTTCTACGACCTGCTCTAGGT..GGGCATTGCGGCCTCC 1356

|| | || | ||| || || | | | | | || ||| |2265 ACGCTCGCTTATATGTACATACTACTATCGTTACTAGGATACCGGTAGAC 2314

. . . . . . .1357 GCGGTGGACGTGTTCTTTTCTAAGCCATGGAGTGAGTGAGC...AGGTGT 1403

| | || | ||| |||| | | | | | | | ||

. . . . .1404 GAA.................................... 1406

||2365 TAAGGCTTCTGTTTCTTCAAGTCCATGTAAGAAGAT 2400 Above: Nucleic acid sequence of human hchi-a protein (GenBank Accession No.AF212229) Below: Nucleic acid sequence of A.Nidulans chi-a protein (GenBank Accession No.D87063) Table 323.6% identity in 267 aa overlap , 56.9% similarity in 267 aa overlap

110 120 130 140 150 160h.pep PVWLQLKRRGREMFEVTGLHDVDQGWMRAVRKHAKGLHIVPRLLFEDWTYD-DFRNVLDS

|+| + |+++ | + |||+ +| | +a.pep SDIEKHYPTDSWNDTGNNVYGCVKQLGLLKRQH-RQLKVL--LSIGGWTYSPNFTNGAT

80 90 100 110 120

170 180 190 200 210 220h.pep EDEIEELSKTVVQVAKNQHFDGFVVEVWNQLLSQKRVGLIHMLTHLAEALHQARLLALLV

++ ++ ++++|++++ + |||+ ++ + +|+ + + +| + |||+ |+ +a.pep PENRARFAQTATKLITDLGFDGIDIDWEYPQNDQQAQNYVDLLRRCREALNAAQGQRRFQ

130 140 150 160 170 180

230 240 250 260 270h.pep IPPAITPGTDQLGMFTHKEEEQLAVLDGFSLMTYDY------STAHQ----PG---PNA

+ + |+ | |+ +++ ++++++| || ++||+||| +++|||

190 200 210 220 230 240

                                                                                                   

|++ |+| + +| + +||+||+ +|| + ++ +|

250 260 270 280 290 300

330 340 350 360 370 380h.pep --PRMVWDSQVSEHF-FEYKKSRSGRHVVFYPTLKS--LQVRLELARELGVGVSIWELGQ

|| |++ ++ ++ + |+|++| | |+ + |++ +|+|| |+ ||a.pep ALPRPGATEQLDTNIGASWSYDPSSREMVSYDTVAAADLKAAYIQSRRLG-GAMWWETSA

310 320 330 340 350 360

        390h.pep   GLDYFYDLLa.pep   DKGGKTANKADGSLIGTFVEDVGGVNNLDRTQNAISYPDSQYDNLKAGFPSS

370 380 390 400 410h.pep: amino acid sequence of human hchi-a protein a.pep: amino acid sequence of A.nidulanschi-a protein (GenBank Accession No.BAA35140)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hchi-a protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 157-1338 position among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 157-1338 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 157-1338 position.

4. isolated people hchi-a protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6, it is characterized in that it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. a generation has the method for the polypeptide of people hchi-a protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hchi-a protein-active operationally is connected in expression regulation sequence, form people hchi-a protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 157-1338 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hchi-a;

(3) under the condition that is fit to expressing human hchi-a protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hchi-a protein-active.

9. energy and the described people hchi-a of claim 7 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.

10. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

11. one kind is used for the method whether test sample exists people hchi-a nucleotide sequence, it is characterized in that it comprises with described probe of claim 10 and sample hybridizes, whether detection probes combination has taken place then, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to people hchi-a nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.