CN1316515A - Human liver cancer related cancer suppressing protein and its coding sequence - Google Patents

Abstract

The invention provides a novel human hALC protein expressed in human liver and its coding sequence, the invention also provides a preparation method of the protein and nucleic acid sequence, and a method for detecting human hALC nucleic acid sequence and polypeptide in a sample.

Description

A human liver cancer-related tumor suppressor protein and its coding sequence

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

During the study of acute lymphocytic leukemia (ALL), it was found that about 20% of the cases were closely related to a fusion protein E2a-Pbx1. This fusion protein was expressed in a fibroblast cell line of house mouse, and further research found that E2a-Pbx1 acted on 12 target genes in it. These target genes were named EF-1 to EF-12 (E2a-Pbx1 target gene in fibroblasts1 to 12). (Mol Cell Biol. 1997 Mar; 17(3):1503-12.).

In the present invention, we cloned the homologous gene of the musculus EF-9 gene in human liver. After further research, we found that this new human gene is not only completely homologous to the house mouse EF-9 gene, but also closely related to the occurrence of human liver cancer, so it was named hALC gene (human Anticogene involved in Liver Cancer).

Before the publication of the present invention, there has not been any disclosure or report of the human hALC protein sequence and its nucleic acid sequence mentioned in this patent application.

The first object of the present invention is to provide a new human gene hALC (Genbank Accession No. AY013707), which is a human hALC protein gene.

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

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

The present invention also provides the application of the human hALC protein polypeptide and coding sequence.

In one aspect of the present invention, an isolated DNA molecule is provided, the molecule includes: a nucleotide sequence encoding a polypeptide having human hALC protein activity, and the nucleotide sequence is the same as that obtained from SEQ ID NO.6 The nucleotide sequence of the nucleotide 31-1671 DNA molecule has at least 70% homology; or the nucleotide sequence can be obtained from the nucleotide sequence of SEQ ID NO.6 under moderately stringent conditions The nucleotide sequence at position 31-1671 hybridizes. 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 31-1671 in SEQ ID NO.6.

In another aspect of the present invention, an isolated human hALC protein polypeptide is provided, which includes: a polypeptide having the amino acid sequence of SEQ ID NO.7, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof things. 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, a method for producing a polypeptide having human hALC protein activity is also provided, the steps of which are as follows:

(1) The nucleotide sequence encoding the polypeptide having human hALC protein activity is operably connected to the expression control sequence to form a human hALC protein expression vector, and the nucleotide sequence and SEQ ID NO.6 are derived from nucleosides The nucleotide sequence at positions 31-1671 of acid has at least 70% homology;

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

(3) cultivating the recombinant cells in step (2) under conditions suitable for expressing the human hALC protein polypeptide;

(4) Isolating a polypeptide having human hALC protein activity.

Preferably, the nucleic acid sequence used in the method has the sequence of positions 31-1671 in SEQ ID NO.6.

The invention also provides an antibody specifically binding to the hALC 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 hALC protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human hALC protein activity, such as the 31-1671 nucleotide sequence in SEQ ID NO.6 and its degenerate sequence. 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 31-1671 nucleotides of the coding frame of the sequence of SEQ ID NO.6 . Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% of the 31-1671 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 31 to 1671 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 31-1671 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 hALC. 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 hALC protein or polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.7 having human hALC protein activity. The term also includes variant forms of the sequence of SEQ ID NO. 7 that have the same function as native human hALC. 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 hALC protein.

Variant forms of the human hALC polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with human hALC DNA under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human hALC polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human hALC polypeptides or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human hALC 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 hALC polypeptide sequence. 100 consecutive amino acids.

In the present invention, "human hALC 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 or similar in nature Amino acids are substituted 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 hALC proteins or polypeptides. The difference between these analogs and the natural human hALC polypeptide may be the difference in the amino acid sequence, or the difference in the 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 hALC polypeptide of the present invention, the human hALC coding sequence can be operably linked to the expression control sequence, thereby forming a human hALC 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 hALC, 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 for human hALC. For example, purified human hALC gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human hALC or antigenic fragments thereof 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 antibodies of the present invention include antibodies that can suppress the function of human hALC, and can also be antibodies that do not affect the function of human hALC. Each type of antibody can be produced by immunizing fragments or functional domains of human hALC gene products, and human hALC gene products and fragments thereof can be produced by recombinant methods or synthesized with a polypeptide synthesizer. Antibodies that bind unmodified forms of human hALC gene products can be obtained by immunizing animals with gene products 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 hALC antibody of the present invention can be used to identify cells expressing human hALC protein or polypeptide, such as Jurkat T cells. For example, a human hALC-specific antibody can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and the human hALC-specific antibody can be exposed to a cell sample and detected by fluorescence microscopy or flow cytometry. Cells bound by human hALC-specific antibodies.

In addition to detecting human hALC 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 and dissolved in a lysis buffer containing detergent. Cell extracts were then separated by SDS polyacrylamide gel electrophoresis (with purified human hALC polypeptide as a positive control) and transferred to nitrocellulose by electrophoretic hybridization. For immunodetection of human hALC polypeptides 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 hALC gene products can also be analyzed by Northern blot technique, that is, the presence and quantity of RNA transcripts of human hALC in the cells can be analyzed.

Northern blot analysis of human hALC DNA and Western blot analysis of human hALC-specific antibodies can be used in combination to confirm the expression of human hALC in biological samples. Human hALC 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, which generally has 8-100 consecutive nucleotides of the human hALC nucleotide coding sequence, preferably 15-50 consecutive nucleotides acid. The probe can be used to detect whether there is a nucleic acid molecule encoding human hALC in a sample.

The present invention also provides a method for detecting whether there is human hALC 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 hALC 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 hALC nucleotide sequence and amino acid sequence of the present invention, human hALC homologous genes or homologous proteins can be screened on the basis of nucleic acid homology or expressed protein homology.

In order to obtain the dot array of human cDNAs or genomic DNAs related to human hALC genes, human cDNA or genomic DNA libraries can be screened with DNA probes, which are all or part of human hALC with ³² P under low stringency conditions obtained by radioactive labeling. The most suitable cDNA library for screening is that from human liver. 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 hALC.

Screening of human hALC homologues based on nucleotide similarity can be accomplished as follows. Human liver cDNA libraries such as Clontech Cat.#74XX (Clontech, Palo Alto, Cal.) can be screened using a randomly primed DNA probe containing all or part of the human hALC gene sequence. To complete the identification of clones with DNA insertion sequences at least 70% homologous to human hALC sequences, use a hybridization solution at a hybridization temperature of 55°C, followed by washing with 0.5×SSC and 0.1% SDS. The cloned DNA insert sequence identified in this way can be further assessed for similarity to the human hALC 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 hALC homologues can also be identified with antibodies against human hALC proteins or polypeptides. For example, commercially available or constructed expression libraries derived from cells or tissues such as liver 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 hALC 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 hALC gene. Tissue expression profiles of newly identified genes can be analyzed similarly as described above.

The full-length human hALC 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 completely possible to obtain the nucleic acid sequence encoding the human hALC 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.

Utilizing the human hALC protein of the present invention, through various conventional screening methods, substances interacting with human hALC, or receptors, inhibitors or antagonists can be screened out.

When the human hALC 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 changed 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 hALC 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 hALC 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 hALC protein polypeptide of the present invention is used as a drug, 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 dosage 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 the homology comparison (FASTA) table of the nucleotide sequences (GenBank Accession No. U72678) of the human hALC protein of the present invention and the EF-9 protein of the house mouse.

Table 3 is the homology comparison (FASTA) table of the amino acid sequence (GenPept Accession No. AAB51038) of the human hALC protein of the present invention and the musculus EF-9 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 is usually according to conventional 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 hALC gene

1. Tissue isolation (liver isolation)

The livers were obtained from 5 normal adult male donors. The livers were removed within four hours after death and immediately placed in liquid nitrogen for cryopreservation.

2. 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, and then the fragments were separated. 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) for packaging, the host bacteria used XL1-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 ABI377 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. Gene full-length cloning (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 hALC gene can usually be obtained.

(2) 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 perform long-length cDNA in the human liver Marathon-Ready cDNA library (Clontech Lab, Inc, USA) distance from the PCR reaction. 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 liver total RNA library. 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 hALC 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'-TCCAGTGTTTCTGGCAGTTG-3' (SEQ ID NO.4) as the forward primer, oligonucleotide R2: 5' -TTGTGGTCTGATTGCACCAT-3'(SEQ ID NO.5) was used as a reverse primer, and the total RNA of the liver was used as a template for RT-PCR amplification. The PCR conditions for R1/R2 were 94°C for 5 minutes, followed by 94°C for 30 35 cycles of 30 seconds at 60°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 1988bp. 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 hALC gene:

The novel human hALC full-length cDNA of the present invention (GenBank Accession No.AY013707. It has not been disclosed before this application) has a length of 1988bp, and the detailed sequence is shown in SEQ ID NO.6, wherein the open reading frame is located at 31-1671 nucleotides . The amino acid sequence of human hALC was deduced according to the full-length cDNA, with a total of 546 amino acid residues, a molecular weight of 59987.88, and a pI of 5.49. See SEQ ID NO.7 for the detailed sequence.

The full-length cDNA sequence of hALC and its encoded protein were used to perform nucleotide and protein homology in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using the BLAST program After searching, it was found that it has a certain homology with the EF-9 gene of the house mouse. On the nucleotide level, it has 86.4% identity (attached table 2) with the mRNA full coding sequence (GenBank Accession No.U72678) of the house mouse EF-9 gene, and on the amino acid level, it has the same The amino acid residues 1-154 of the 9 protein (GenPept Accession No. AAB51038) have 92.9% identity and 92.0% similarity (Supplementary Table 3). It can be seen from the above that the hALC gene and the house mouse EF-9 gene have high homology in both nucleic acid and protein levels, and it can be considered that the two also have high similarity in function.

In addition to being used as a member of the family for further functional research, the human hALC of the present invention can also be used to produce fusion proteins with other proteins, such as immunoglobulins to produce fusion proteins. In addition, the human hALC 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 hALC protein of the present invention is exchanged with the N-terminal of the murine EF-9 protein to produce a new protein with higher activity or new properties.

The antibody against human hALC 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 hALC nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human hALC or inhibit the overexpression of human hALC. The human hALC protein or its active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by the absence, non-function or abnormality of human hALC. 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 hALC protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or lower side effects (e.g. less or no immunogenicity in humans).

Example 3

Functional Study of Human hALC Protein

1. Expression of EF-9 gene in house mouse:

Experimental evidence shows that the EF-9 gene of the house mouse is only expressed specifically in the liver in the house mouse. After further research, we found that the expression of the EF-9 gene in the house mouse begins in the late embryonic development of the house mouse.

2. Expression of human hALC gene:

By studying the expression of hALC gene in a large number of human tissues, we found that hALC gene is also specifically expressed only in human liver. However, in HCC tissues, hALC gene expression was lost.

analyze:

Through comparative analysis of the above experimental results, we can draw a conclusion: hALC gene may be related to tissue differentiation, is a potential marker for liver cancer disease detection, and is expected to be applied in gene therapy.

Example 4

Preparation and purification of human hALC polypeptide

In this example, the full-length human hALC 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 hALC 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 of human hALC (SEQ ID NO.6), design primers to 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 were introduced into the anti-primers (this depends on the pGEX-2T vector selected), so as to construct the expression vector. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hALC 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-hALC containing the pGEX-2T-hALC expression vector was screened and identified.

Isolation and Identification of Engineering Bacteria Expressing GST-hALC Recombinant Protein

Pick the DH5α-pGEX-2T-hALC engineering bacteria of a single colony in 3 ml of LB medium containing 100 μg/ml ampicillin and culture overnight with shaking, and draw the culture solution into new LB medium (containing 100 μg/ml ampicillin) at a concentration of 1:100. /ml ampicillin) for about 3 hours, and when the OD ₆₀₀ reached 0.5, IPTG was added to a final concentration of 1 mmol/L to continue culturing at 37°C for 0, 1, 2, and 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-hALC fusion protein.

Extraction and purification of GST-hALC fusion protein

The engineering bacteria DH5α-pGEX-2T-hALC expressing GST-hALC fusion expression protein were 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-hALC Sepharose4B, 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 60kDa is human hALC protein.

Example 5

Eukaryotic expression of human hALC protein or polypeptide in insect cells

1. Construction of Human hALC Baculovirus Expression Vector and Transfection to Sf9 Insect Cell Line

According to the full-length coding sequence of human hALC (SEQ ID NO.6), design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the forward and reverse primers respectively (this may vary depending on the carrier selected). ) to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hALC cDNA 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 culture 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 with recombinant expression vector

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 placed in the anti- Human hALC antibody solution was blocked for 1 hour, TBS solution was shaken and washed for 5 minutes, a total of 2 times, and then the membrane was placed in biotin-labeled anti-human hALC primary antibody secondary antibody solution, shaken for 1 hour, TBS washed, added React the avidin-alkaline phosphatase complex 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 hALC were picked.

3. Extraction and purification of human hALC protein

The supernatant of Sf9 cell clones highly expressing human hALC was used to infect a large number of Sf9 cells. After 48 hours of infection, the cells were collected 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, 1μg per 2×10 8 cells /ml pepsin inhibitor, 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 hALC protein was detected by SDS-PAGE electrophoresis. The protein band at 60kDa is human hALC protein.

Example 6

Preparation of anti-human hALC antibody

1. Preparation of immunized mice and splenocytes: The human hALC protein obtained in Example 5 and Example 6 was separated by chromatography and then used for later use, or it could be separated by SDS-PAGE gel electrophoresis, and the electrophoresis band was separated from 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. Feeder cells were prepared according to the method on page 371 of "Tissue Culture and Molecular Cytological Techniques" (supra).

3. Cell fusion was carried out according to the method on page 213 of "Tissue Culture and Molecular Cytology Techniques" (supra).

4. Antibody detection: After 10-15 days of cell fusion, it is necessary to check each hole. Once vigorous hybrid cell colony growth is found, human hALC protein is used for preliminary screening of antibody activity. Commonly used methods include: immunofluorescence test, emission immunoassay assay (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) Sequence features:

(A) Length: 50bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

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

(2) Information of SEQ ID NO.2

(i) Sequence features:

(A) Length: 13bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(iii) Sequence description: SEQ ID NO.2

AATTCGGCAC GA G 13

(3) Information of SEQ ID NO.3

(i) Sequence features:

(A) Length: 9bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(iii) Sequence description: SEQ ID NO.3

GCCGTGCTC 9(4) Information on SEQ ID NO.4 (ⅰ). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (Ⅲ). Sequence description: SEQ ID NO.4TCCAGTGTTTCTGGCAGTTG 20(5) Information of SEQ ID NO.5 (ⅰ). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (Ⅲ). Sequence description: SEQ ID NO.5TTGTGGTCTGATTGCACCAT 20(6) Information of SEQ ID NO.6 (ⅰ) Sequence characteristics:

(A) Length: 1988bp

(B) Type: Nucleotide

(C) chain: single chain

(D)拓扑结构：线性(ⅱ)分子类型：核苷酸(ⅲ)序列描述：SEQ ID NO.61 TCCAGTGTTT CTGGCAGTTG GTCCAGAAGG ATGCCTCCAT TCCTGCTTCT51 CACCTGCCTC TTCATCACAG GCACCTCCGT GTCACCCGTG GCCCTAGATC101 CTTGTTCTGC TTACATCAGC CTGAATGAGC CCTGGAGGAA CACTGACCAC151 CAGTTGGATG AGTCTCAAGG TCCTCCTCTA TGTGACAACC ATGTGAATGG201 GGAGTGGTAC CACTTCACGG GCATGGCGGG AGATGCCATG CCTACCTTCT251 GCATACCAGA AAACCACTGT GGAACCCACG CACCTGTCTG GCTCAATGGC301 AGCCACCCCC TAGAAGGCGA CGGCATTGCG CAACTGCCAG GCTTGTGCCA351 CGCTTCAATG GGAACTGCTG TCTCTGGAAC ACCACGGTGG AAGTCAAGGC401 TTGCCCTTGG AGGCTACTAT GTGTATCGTC TGACCAAGCC CAGCGTCTGC 451 TTCCACGTCT ACTGTGGTCA TTTTTATGAC ATCTGCGACG AGGACTGCCA501 TGGCAGCTGC TCAGATACCA GCGAGTGCAC ATGCGCTCCA GGAACTGTGC551 TAGGCCCTGA CAGGCAGACA TGCTTTGATG AAAATGAATG TGAGCAAAAC601 AACGGTGGCT GCAGTGAGAT CTGTGTGAAC CTCAAAAACT CCTACCGCTG651 TGAGTGTGGG GTTGGCCGTG TGCTAAGAAG TGATGGCAAG ACTTGTGAAG701 ACGTTGAAGG ATGCCACAAT AACAATGGTG GCTGCAGCCA CTCTTGCCTT751 GGATCTGAGA AAGGCTACCA GTGTGAATGT CCCCGGGGCC TGGTGCTGTC801 TGAGGATAAC CACACTTGCC AAGTCCCTGT GTTGTGCAAA TCAAATGCCA851 TTGAAGTGAA CATCCCCAGG GAGCTGGTTG GTGGCCTGGA GCTCTTCCTG901 ACCAACACCT CCTGCCGAGG AGTGTCCAAC GGCACCCATG TCAACATCCT951 CTTCTCTCTC AAGACATGTG GTACAGTGGT CGATGTGGTG AATGACAAGA1001 TTGTGGCCAG CAACCTCGTG ACAGGTCTAC CCAAGCAGAC CCCGGGGAGC1051 AGCGGGGACT TCATCATCCG AACCAGCAAG CTGCTGATCC CGGTGACCTG1101 CGAGTTTCCA CGCCTGTACA CCATTTCTGA AGGATACGTT CCCAACCTTC1151 GAAACTCCCC ACTGGAAATC ATGAGCCGAA ATCATGGGAT CTTCCCATTC1201 ACTCTGGAGA TCTTCAAGGA CAATGAGTTT GAAGAGCCTT ACCGGGAAGC1251 TCTGCCCACC CTCAAGCTTC GTGACTCCCT CTACTTTGGC ATTGAGCCCG1301 TGGTGCACGT GAGCGGCTTG GAAAGCTTGG TGGAGAGCTG CTTTGCCACC1351 CCCACCTCCA AGATCGACGA GGTCCTGAAA TACTACCTCA TCCGGGATGG1401 CTGTGTTTCA GATGACTCGG TAAAGCAGTA CACATCCCGG GATCACCTAG1451 CAAAGCACTT CCAGGTCCCT GTCTTCAAGT TTGTGGGCAA AGACCACAAG1501 GAAGTGTTTC TGCACTGCCG GGTTCTTGTC TGTGGAGTGT TGGACGAGCG1551 TTCCCGCTGT GCCCAGGGTT GCCACCGGCG AATGCGTCGT GGGGCAGGAG1601 GAGAGGACTC AGCCGGTCTA CAGGGCCAGA CGCTAACAGG CGGCCCGATC1651 CGCATCGACT GGGAGGACTA GTTCGTAGCC ATACCTCGAG TCCCTGCATT1701 GGACGGCTCT GCTCTTTGGA GCTTCTCCCC CCACCGCCCT CTAAGAACAT1751 CTGCCAACAG CTGGGTTCAG ACTTCACACT GTGAGTTCAG ACTCCCAGCA1801 CCAACTCACT CTGATTCTGG TCCATTCAGT GGGCACAGGT CACAGCACTG1851 CTGAACAATG TGGCCTGGGT GGGGTTTCAT CTTTCTAGGG TTGAAAACTA1901 AACTGTCCAC CCAGAAAGAC ACTCACCCCA TTTCCCTCAT TTCTTTCCTA1951 CACTTAAATA CCTCGTGTAT GGTGCAATCA GACCACAA(7)SEQ ID NO.7的信息

(i) Sequence features:

(A) Length: 546 amino acids

(B) type: amino acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: polypeptide

(ⅲ)序列描述：SEQ ID NO.7:1 MPPFLLLTCL FITGTSVSPV ALDPCSAYIS LNEPWRNTDH QLDESQGPPL51 CDNHVNGEWY HFTGMAGDAM PTFCIPENHC GTHAPVWLNG SHPLEGDGIA101 QLPGLCHASM GTAVSGTPRW KSRLALGGYY VYRLTKPSVC FHVYCGHFYD151 ICDEDCHGSC SDTSECTCAP GTVLGPDRQT CFDENECEQN NGGCSEICVN201 LKNSYRCECG VGRVLRSDGK TCEDVEGCHN NNGGCSHSCL GSEKGYQCEC251 PRGLVLSEDN HTCQVPVLCK SNAIEVNIPR ELVGGLELFL TNTSCRGVSN301 GTHVNILFSL KTCGTVVDVV NDKIVASNLV TGLPKQTPGS SGDFIIRTSK351 LLIPVTCEFP RLYTISEGYV PNLRNSPLEI MSRNHGIFPF TLEIFKDNEF401 EEPYREALPT LKLRDSLYFG IEPVVHVSGL ESLVESCFAT PTSKIDEVLK451 YYLIRDGCVS DDSVKQYTSR DHLAKHFQVP VFKFVGKDHK EVFLHCRVLV501 CGQLGLDERSRC AQGCGRDRSRID

h.seq：人hALC蛋白的核酸序列(GenBank Accession No.AY013707)m.seq：家鼠EF-9蛋白的核酸序列(GenBank Accession No.U72678)Schedule 2

h.seq: Nucleic acid sequence of human hALC protein (GenBank Accession No.AY013707) m.seq: Nucleic acid sequence of murine EF-9 protein (GenBank Accession No.U72678)

h.pep：人hALC蛋白氨基酸序列m.pep：家鼠EF-9蛋白氨基酸序列(GenBank Accession No.AAB51038)Schedule 3

h.pep: amino acid sequence of human hALC protein m.pep: amino acid sequence of house mouse EF-9 protein (GenBank Accession No.AAB51038)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hALC protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 31-1671 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 31-1671 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 31-1671 position.

4. isolated people hALC 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 hALC protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hALC protein-active operationally is connected in expression regulation sequence, form people hALC protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 31-1671 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 hALC;

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

(4) isolate polypeptide with people hALC protein-active.

9. energy and the described people hALC 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 hALC 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 hALC nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.