CN1952130A - Liver cancer high-expression gene PEG10, its encoded protein and use - Google Patents

Abstract

The invention discloses a liver cancer highly expressed gene PEG10. The invention also discloses the coding protein of the PEG10 gene. In addition, the invention also discloses the application of the PEG10 gene as a liver cancer diagnostic marker. The PEG10 gene of the present invention is a highly expressed gene of liver cancer, and the expression rate of PEG10 in liver cancer is as high as 66%, while the expression rate and expression level in liver tissues adjacent to cancer are extremely low. Therefore, PEG10 polynucleotide, PEG10 protein and its antibody, and PEG10 protein-related antagonists and agonists can provide new therapeutic approaches for the treatment of various diseases including liver cancer, and thus have great application prospects.

Description

Highly expressed gene PEG10 in liver cancer, its encoded protein and its application

technical field

The invention relates to the field of biotechnology, in particular, the invention relates to an imprinted gene PEG10 mainly highly expressed in human liver cancer tissues and its encoded protein; in addition, the invention also relates to the application of the PEG10 gene.

Background technique

In 1986, Renato Dulbecco, a famous biologist and Nobel laureate, first proposed the "Human Genome Project" (Human Genomic Project, referred to as HGP) in Science magazine. In October 1990, the U.S. government decided to invest 3 billion U.S. dollars to officially launch the "Human Genome Project". It is expected to obtain the entire gene sequence of the human body (a total of about 3 billion nucleotides in total) by 2005; function, thereby uncovering the mystery of all human genetic information, and making human beings' understanding of themselves reach a new height.

The Human Genome Project can be said to be one of the greatest centuries-old projects for human beings to understand themselves. At the end of 1997, someone proposed that the ultimate goal of HGP should be to provide a biological periodic table, the "elements" of this periodic table are the 100,000-140,000 genes that determine all human traits. Completing the sequencing of 3 billion bases only lays the structural foundation for this goal (i.e., structural genomics), while comparative genomics (including its encoded proteins) and functional studies of the entire genome (functional genomics) in the genome It can directly play its important role in the discovery of value. With the development of genomics, people have designed and created many important biomolecules, which are widely used in various fields such as pharmaceuticals, agriculture, food, chemical industry, cosmetics, environment, energy, etc., which will not only promote the progress of science, but also produce amazing Economic benefits, thus triggering the industrial revolution, the life science industry based on genomics has been formed. The limited genetic resources of human beings are being allocated at one time, and the companies with the highest gene efficiency and the largest number are expected to use their gene patents to monopolize the future biological and pharmaceutical industry markets. Bill Gates once said: The next person who creates greater wealth will appear in the field of genes.

With the rapid development of the genome project, it will become a reality in the near future to conquer malignant tumors including liver cancer. my country is an area with a high incidence of primary hepatocellular carcinoma (HCC), and its mortality rate has accounted for the first and second in some rural areas and cities in my country. It is a major disease that seriously affects the health of our people. There are 386,000 people in the world people die from liver cancer, and 45% of them are in China. At present, it is believed that the occurrence and development of liver cancer, like other malignant tumors, is a complex process involving multiple genes and factors, including somatic gene mutation, loss of tumor suppressor genes, activation and overexpression of oncogenes, etc. The abnormal activation or inactivation of oncogenes, tumor suppressor genes, and related genes is the molecular basis of liver cancer. Theoretically, more genes should be involved, but the detailed functions of many genes that have been discovered so far are still not very clear. Oncogenes or abnormally expressed genes that have been proven to be related to human liver cancer include; met)/HGF, c-fins(CSF-IR), c-erbB-1(EGF-R)/c-erbB-2(neu), Ras, Raf, c-myc, c-ets-2, etc., However, there is no specific expression gene for liver cancer. Therefore, it is not only important to find new abnormally expressed or deleted genes in liver cancer, understand the molecular mechanism of growth, differentiation, regeneration and carcinogenesis of liver cells from the gene level, and clarify the signal transmission process and pathway. It has important biological significance, and has great clinical application significance and economic development value for early gene diagnosis and information therapy of liver cancer, and can also occupy a new commanding height in the research of human genome.

With the rapid development of molecular biology techniques, new methods for finding and isolating genes are constantly emerging. There are four main methods: 1. Methods starting from eDNA or mRNA: using the differences in gene expression in cells to isolate disease-causing genes is a class of An important method, currently progressing rapidly. Including cDNA subtractive hybridization method, mRNA differential display technology, EST (Expressed sequence tags) differential hybridization screening, etc.; 2. Starting from the analysis of proteins; starting from the position, binding characteristics or partial functions of proteins, first isolate the desired protein, and then obtain the corresponding gene. Including immunoprecipitation, two-dimensional electrophoresis, yeast two-hybrid system method, etc.; 3. Starting from the search for genomic DNA fragments: find genetic markers or partial functional information closely linked to the target gene locus, so as to determine the presence of genes related to a trait in the genome The position of a segment in the gene, and then use different methods to find the expression sequence in the chromosome of the segment, and finally clone the target gene. Including complex probe library screening method, Northern hybridization method, splice site screening method, CpG island screening method, interspecies homologous sequence hybridization method, PCR direct selection method, exon capture, consensus sequence cloning method, etc.; 4. From the whole Starting from the genome: directly isolate the clone of the gene based on the effect of the gene, without knowing its biochemical function or map location in advance, and without assuming the number of genes or their interaction mode, including genome mismatch scanning and representative difference analysis, etc. . The above-mentioned methods for finding new genes have their own advantages and disadvantages. Which method to use should be based on the conditions and advantages of each laboratory, and adopt a practical method to maximize strengths and avoid weaknesses.

In 1992, American scholar Liang Peng et al. first applied mRNA differential display technology to separate and identify gene expression in different tissues and cells. Using this method, people have discovered a large number of unknown genes. For example, Liang et al. used this method to compare normal breast and breast cancer The mRNA of novel gene expressed in multiple cancers, found a full-length 600bp S1 gene: Chen et al. The new gene N8, 2.4kb in length, is located on chromosome 8q13 and is overexpressed in lung cancer tissues; Japanese scholars Shibabara et al. , 166(2):297-301) used this method to isolate a new gene MA-3 that causes programmed cell death, and so on. However, there are still some deficiencies in DDPCR technology. Although some scholars have made many improvements in this area in recent years, such as the rationality of primer design and how to reduce false positives, etc., they still need to be continuously improved. It will be a very useful tool in the field of life sciences.

Therefore, it is of great significance to study and develop genes and/or proteins highly expressed in liver cancer for therapeutic and diagnostic purposes. There is an urgent need in the art for new genes and/or proteins that are highly expressed in liver cancer.

The imprinted gene PEG10, localized on human chromosome 7q21, PEG10 is located near the SGCE gene, and its mouse counterpart has recently been shown to be imprinted. Therefore, there may be a new imprinted gene group in human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 are homologous to several vertebrate proteins. Placental PEG10 is hyporegulated during the early hypoxic phase and hyperactive during 11-12 weeks of pregnancy. Exogenous expression of PEG10 contributes to tumorigenic activity. PEG10 protein associates with mediator SIAH1, and overexpression of PEG10 reduces mediator SIAH1 cell death. The structure shows that the imprinted gene PEG10 plays an important role in liver regeneration and tumor suppression of human hepatocytes.

In view of the important functions of the imprinted gene PEG10, it is of great significance to research and develop the imprinted gene PEG10.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide an imprinted gene PEG10 (Paternally Expressed 10) highly expressed in liver cancer.

The second technical problem to be solved by the present invention is to provide a human protein PEG10.

The third technical problem to be solved by the present invention is to provide the application of PEG10 gene as a diagnostic marker for liver cancer.

The present invention obtains a gene fragment from liver cancer tissue, and then clones a full-length cDNA sequence of a new gene positively correlated with PEG10 through gene cloning technology, and the gene sequence is highly related to primary hepatocellular carcinoma. What is interesting is that the expression of this gene is as high as 66% in liver cancer, while the expression in paracancerous liver tissue is not or very low. The gene encodes a 568 amino acid protein located in the cytoplasm.

In order to solve the above technical problems, the present invention is realized through the following technical solutions:

In one aspect of the present invention, a kind of isolated DNA molecule is provided, and this molecule comprises: the nucleotide sequence of coding the polypeptide with human PEG10 protein activity, described nucleotide sequence and SEQ ID NO.1 from The nucleotide sequence of the nucleotide 1-1707 DNA molecule has at least 70% homology; or the nucleotide sequence can be obtained from nucleotides in SEQ ID NO.1 under moderately stringent conditions The nucleotide sequence at position 1-1707 hybridizes. Preferably, the sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.2. More preferably, said sequence has the nucleotide sequence from nucleotide 1-1707 in SEQ ID NO.1.

In another aspect of the present invention, an isolated human PEG10 protein polypeptide is provided, which includes: a polypeptide having an amino acid sequence of SEQ ID NO.2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof thing. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.2.

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 another aspect of the present invention, a method for preparing a polypeptide having human PEG10 protein activity is also provided, the method comprising:

(1) The nucleotide sequence encoding the polypeptide having human PEG10 protein activity is operably connected to the expression control sequence to form a human PEG10 protein expression vector, and the nucleotide sequence and SEQ ID NO.1 are derived from nucleosides The nucleotide sequence of acid 1-1707 has at least 70% homology;

(2) transferring the expression vector in step (1) into a host cell to form a recombinant cell of human PEG10 protein;

(3) cultivating the recombinant cells in step (2) under conditions suitable for expressing the human PEG10 protein polypeptide;

(4) Isolating a polypeptide having human PEG10 protein activity.

Preferably, the nucleic acid sequence used in the method has the sequence of positions 1-1707 in SEQ ID NO.1.

The invention also provides an antibody specifically binding to the PEG10 protein polypeptide.

In the present invention, "isolated" DNA means that the DNA or fragment has been separated from the sequences flanking it in the natural state, and also means that the DNA or fragment has been separated from the components accompanying the nucleic acid in the natural state, And has been separated from the proteins that accompany it in the cell.

In the present invention, the term "human PEG10 protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human PEG10 protein activity, such as the 1-1707th nucleotide sequence in SEQ ID NO.1 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 nucleotides 1-1707 of the coding frame of the sequence of SEQ ID NO.1 . Due to the degeneracy of codons, the degenerate sequence with a homology as low as about 70% of the 1-1707 nucleotide sequence in SEQ ID NO.1 can also encode the sequence described in SEQ ID NO.2 . The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 1-1707 in SEQ ID NO.1 under moderately stringent conditions, preferably under highly stringent conditions. Wherein, "stringent conditions" refers to the membrane washing conditions after the nucleotide sequence is hybridized on the membrane. For example, in this field, about 150ml of washing liquid can be poured into the hybridization tube for washing the membrane with low stringency, put into the hybridization membrane, and shake continuously at room temperature for about 20 minutes, while the washing membrane with high stringency can be poured into the hybridization tube. Put about 200ml of washing solution into the hybridization membrane, and shake continuously for about 20 minutes in a shaker at 50°C. 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 1-1707 in SEQ ID NO.1 At least 95% nucleotide sequence.

The term also includes variants of the open reading frame sequence of SEQ ID NO. 1 that encode a protein having the same function as human PEG10. 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, the term "human PEG10 protein or polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.2 having PEG10 protein activity. The term also includes variant forms of the sequence of SEQ ID NO. 2 that have the same function as native human PEG10. 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 the PEG10 protein.

Variant forms of human PEG10 polypeptides of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, encoded by DNA that can hybridize with human PEG10 DNA under high or low stringency conditions Proteins, and polypeptides or proteins obtained using antiserum against human PEG10 polypeptides. The present invention also provides other polypeptides, such as fusion proteins comprising human PEG10 polypeptide or fragments thereof. In addition to substantially full-length polypeptides, the present invention also includes soluble fragments of human PEG10 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 PEG10 polypeptide sequence. 100 consecutive amino acids.

In the present invention, "human PEG10 conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO.2, 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 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 PEG10 protein or polypeptide. The difference between these analogues and the natural human PEG10 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 PEG10 polypeptide of the present invention, the PEG10 coding sequence can be operably linked to the expression control sequence, thereby forming a human PEG10 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 specific to human PEG10, 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 PEG10. For example, the purified human PEG10 gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human PEG10 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 antibody of the present invention includes an antibody that can inhibit the function of human PEG10, or an antibody that does not affect the function of human PEG10. Each type of antibody can be produced by immunizing fragments or functional domains of human PEG10 gene products, and human PEG10 gene products and fragments thereof can be produced by recombinant methods or synthesized with a polypeptide synthesizer. Antibodies that bind unmodified forms of the PEG10 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 PE610 antibody of the present invention can be used to identify cells expressing human PEG10 protein or polypeptide, such as Jurkat T cells. For example, a PEG10-specific antibody can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and the PEG10-specific antibody can be exposed to a cell sample, and the PEG10-specific antibody can be detected by fluorescence microscopy or flow cytometry. Cells to which specific antibodies bind.

In addition to detecting human PEG10 on the cell surface, the protein can also be analyzed by Western blot. Cell lysates can be extracted from cultured cells or tissue samples from patients such as adrenal glands and dissolved in lysis buffer containing detergents. Cell extracts were then separated by SDS polyacrylamide gel electrophoresis (with purified human PEG10 polypeptide as a positive control) and transferred to nitrocellulose by electrophoretic hybridization. For immunodetection of PEG10 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 the PEG10 gene product, ie, the presence and quantity of RNA transcripts of human PEG10 in the cells, can also be analyzed using Northern blot techniques.

Northern blot analysis of human PEG10 DNA and Western blot analysis with human PEG10-specific antibody can be used in combination to confirm the expression of human PEG10 in biological samples. Human PEG10 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.

The full-length human PEG10 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 PEG10 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.

Utilizing the human PEG10 protein of the present invention, substances or substances that interact with human PEG10, such as receptors, inhibitors or antagonists, can be screened out through various conventional screening methods.

When the human PEG10 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.

The invention also provides the application of PEG10 gene as a diagnostic marker for liver cancer. The PEG10 gene of the present invention is a highly expressed gene of liver cancer, and the expression rate of PEG10 in liver cancer is as high as 66%, while the expression rate and expression level in liver tissues adjacent to cancer are extremely low. Therefore, the PEG10 gene can be used as a diagnostic marker for liver cancer, making in-depth research on it possible to quantify the detection indicators of liver cancer and contribute to further clinical promotion. In addition, PEG10 polynucleotides, PEG10 proteins and their antibodies, and PEG10 protein-related antagonists and agonists can provide new therapeutic approaches for the treatment of various diseases including liver cancer, and thus have great application prospects.

Description of drawings

Figure 1 is a graph showing the expression of the imprinted gene PEG10 of the present invention in liver cancer/para-cancer tissues verified by RT-PCR, N is the para-cancer tissue, C is the cancer tissue, and β-actin is used as an internal control.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. 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, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Example 1

1. Tissue isolation

Surgical patients derived from HBV-positive primary liver cancer expressing alpha-fetoprotein (RT-PCR confirmed that AFP was positive in liver cancer and negative in adjacent tumors). Once the surgically resected liver was isolated from the body, the lesion and surrounding paracancerous tissues 5 cm away were quickly excised and stored in liquid nitrogen (-80°C). The diagnosis of cancer and paracancer is based on pathological diagnosis.

2. Total RNA extraction kit

The RNA extraction reagent uses TRIzol reagent (GIBCO/BRL), which is produced based on the one-step extraction method with acidic phenol. The utensils and water used for RNA extraction must be RNase-free to ensure an RNase-free environment in the experiment.

3. RNA extraction steps

Dry-bake the pestle, homogenizer and other utensils at 200°C for 4 hours to remove RNase and cool down; add liquid nitrogen to pre-cool, take the tissue out of the liquid nitrogen quickly, and grind it into powder; use a spatula to put the tissue into the pre- Add TRIzol reagent to the homogenizer and homogenize for several minutes; transfer the homogenized liquid into an RNase-free centrifuge tube, add chloroform, and centrifuge at 4°C to separate layers; transfer the upper aqueous phase to an RNase-free In a centrifuge tube, add isopropanol and centrifuge at 4°C to precipitate RNA; wash the precipitate twice with 75% ethanol; dissolve the precipitate with RNase-free deionized water. Quality identification of the extracted RNA: measure the ratio of 260/280 (both ratios are 1.7 to 2.0) with an ultraviolet spectrophotometer; and observe whether there is degradation in MOPS formaldehyde denaturing gel.

4. Synthesis of cDNA

Take 2 μg of total RNA, add 1 μL of OligodT ₁₆ , incubate at 70°C for 3 minutes, and immediately denature on ice for 5 minutes. Add 5× buffer, 2 μL each of DTT and 50 mg/L dNTP, and 1 μL reverse transcriptase, mix well, and keep at 42°C for 2 hours. The final concentration of the template is usually 1 μg/100 μL.

5. RT-PCR amplification

Design primers, PEG10: forward primer is 5'-TGCTTCTGGCAACTTCATTG-3', backward primer is 5'-TCAAATGACAGCACCTCTCG-3'; Beta-actin: forward primer is 5'-TCACCCACACTGTGCCCATCTACGA-3', backward primer is 5''-CAGCGGAACCGCTCATTGCCAATGG-3'. Using β-actin as internal control, the components in the reaction mixture are: β-actin(F), β-actin(R), PEG10(F), PEG10(R), 10×Buffer, MgCl ₂ , dNTP, Taq DNA The polymerase and cDNA template were respectively 0.2, 0.2, 0.4, 0.4, 1.0, 1.0, 0.2, 0.1 and 5 μL cDNA template, and finally ddH ₂ O was added to make the reaction system 10 μL. The reaction conditions of PCR were denaturation at 94°C for 5 minutes; then each cycle was 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s; finally, extension at 72°C for 7 min.

6. Results

The results are shown in Figure 1: using RT-PCR technology to detect the expression difference of PEG10 gene in 32 pairs of liver cancer and adjacent tissues, it was found that 24 of the 32 cases had significantly increased PEG10 gene, that is, the expression level of PEG10 gene in liver cancer tissue The expression rate is as high as 66%.

Example 2

Preparation and purification of human PEG10 polypeptide

In this example, the full-length PEG10 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 PEG10 polypeptide in the form of GST fusion protein in Escherichia coli.

1. Construction of prokaryotic expression vector and transformation of Escherichia coli

According to the full-length coding sequence of human PEG10 (SEQ ID NO.1), 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 PEG10 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-PEG10 containing the pGEX-2T-PEG10 expression vector was screened and identified.

2. Isolation and identification of engineering bacteria expressing GST-PEG10 recombinant protein

Pick the DH5α-pGEX-2T-PEG10 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 a 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-PEG10 fusion protein.

3. Extraction and purification of GST-PEG10 fusion protein

The engineering bacteria DH5α-pGEX-2T-PEG10 expressing GST-PEG10 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 complete ultrasonic liquid, shake at 37°C for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes to precipitate glutathione bound to GST-PEG10 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 31kDa is human PEG10 protein.

sequence listing

<110> Shanghai Human Genome Research Center

<120> Liver cancer highly expressed gene PEG10, its encoded protein and its application

<130> NP-10015

<160> 2

<210> 1

<211> 6861

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (1)..(1707)

<400> 1

1 ATGTGCCACA AACTGACGGA TGGGCGCTTC AGTGGCGCTA CAATCCAGAA CGAGGAGCAG

61 CGATATAGGA GCTGCAGACA GAAAACTGAA TTGCGCTTTA CATGCGCTGC AAAATCTGAG

121 CTGCAGAAGA GTAGCACTAC AAAAAACGCT ACAAAAACGA TGCGATTAGC CAGCCCGACA

181 ACAGCGCTTC ATTGCGCTGC GAGGCACATG AACTGCAGAG CGCCTCCTCC TACAGCGCCT

241 CCTCAGAGCC GCCTCCCCTG CAGCGCCGTG ACCCAGAGTC CTTGTGCTAC CATGCGGCCT

301 TCGCAGGCCC GTGCGCCCAG CTCGAGAGCA CGTGACTTCT GGATCCAGGA CCATCGGTCT

361 GAGGCCCCGC ATGAGTTAGT AGGGAGGGGT AGTGCCCTGC TAGTGTCGGG GCGCCATGGT

421 GATGTTCGGA GGGCGCAGCA CGCAGACCAG GGTTCGGATG GGACGCTGTG GTGTCTTTTT

481 GGGTCGGTCG GGTGGTATAG GGCGATAAAT AAGCGGGTTT TGAAAACAAA AAAAAGAAGG

541 AGTGGAAGAG GGGGCCAGGA TCCAGGCCTC CATCCCCACA GAAGTGAAGC TACAGCTGGG

601 AGGTCTCCTC CCACCCCAAC CGTCACCCTG GGTCCCGACT GCCCACCTCC TCCTCCTCCC

661 CCTCCCCCCA ACAACAACAA CAACAACAAC TCCAAGCACA CCGGCCATAA GAGTGCGTGT

721 GTCCCCAACA TGACCGAACG AAGAAGGGAC GAGCTCTCTG AAGAGATCAA CAACTTAAGA

781 GAGAAGGTCA TGAAGCAGTC GGAGGAGAAC AACAACCTGC AGAGCCAGGT GCAGAAGCTC

841 ACAGAGGAGA ACACCACCCT TCGAGAGCAA GTGGAACCCA CCCCTGAGGA TGAGGATGAT

901 GACATCGAGC TCCGCGGTGC TGCAGCAGCT GCTGCCCCAC CCCCTCCAAT AGAGGAAGAG

961 TGCCCAGAAG ACCTCCCAGA GAAGTTCGAT GGCAACCCAG ACATGCTGGC TCCTTTCATG

1021 GCCCAGTGCC AGATCTTCAT GGAAAAGGC ACCAGGGATT TCTCAGTTGA TCGTGTCCGT

1081 GTCTGCTTCG TGACAAGCAT GATGACCGGC CGTGCTGCCC GTTGGGCCTC AGCAAAGCTG

1141 GAGCGCTCCC ACTACCTGAT GCACAACTAC CCAGCTTTCA TGATGGAAAT GAAGCATGTC

1201 TTTGAAGACC CTCAGAGGCG AGAGGTTGCC AAACGCAAGA TCAGACGCCT GCGCCAAGGC

1261 ATGGGGTCTG TCATCGACTA CTCCAATGCT TTCCAGATGA TTGCCCAGGA CCTGGATTGG

1321 AACGAGCCTG CGCTGATTGA CCAGTACCAC GAGGGCCTCA GCGACCACAT TCAGGAGGAG

1381 CTCTCCCACC TCGAGGTCGC CAAGTCGCTG TCTGCTCTGA TTGGGCAGTG CATTCACATT

1441 GAGAGAAGGC TGGCCAGGGC TGCTGCAGCT CGCAAGCCAC GCTCGCCACC CCGGGCGCTG

1501 GTGTTGCCTC ACATTGCAAG CCACCACCAG GTAGATCCAA CCGAGCCGGT GGGAGGTGCC

1561 CGCATGCGCC TGACGCAGGA AGAAAAAGAA AGACGCAGAA AGCTGAACCT GTGCCTCTAC

1621 TGTGGAACAG GAGGTCACTA CGCTGACAAT TGTCCTGCCA AGGCCTCAAA GTCTTCGCCG

1681 GCGGGAAACT CCCCGGCCCC GCTGTAGAGG GACCTTCAGC GACCGGGCCA GAAATAATAA

1741 GGTCCCCACA AGATGATGCC TCATCTCCAC ACTTGCAAGT GATGCTCCAG ATTCATCTTC

1801 CGGGCAGACA CACCCTGTTC GTCCGAGCCA TGATCGATTC TGGTGCTTCT GGCAACTTCA

1861 TTGATCACGA ATATGTTGCT CAAAATGGAA TTCCTCTAAG AATCAAGGAC TGGCCAATAC

1921 TTGTGGAAGC AATTGATGGG CGCCCCATAG CATCGGGCCC AGTTGTCCAC GAAACTCACG

1981 ACCTGATAGT TGACCTGGGA GATCACCGAG AGGTGCTGTC ATTTGATGTG ACTCAGTCTC

2041 CATTCTTCCC TGTCGTCCTA GGGGTTCGCT GGCTGAGCAC ACATGATCCC AATATCACAT

2101 GGAGCACTCG ATCTATCGTC TTTGATTCTG AATACTGCCG CTACCACTGC CGGATGTATT

2161 CTCCAATACC ACCATCGCTC CCACCACCAG CACCACAACC GCCACTCTAT TATCCAGTAG

2221 ATGGATACAG AGTTTACCAA CCAGTGAGGT ATTACTATGT CCAGAATGTG TACACTCCAG

2281 TAGATGAGCA CGTCTACCCA GATCACCGCC TGGTTGACCC TCACATAGAA ATGATACCTG

2341 GAGCACACAG TATTCCCAGT GGACATGTGT ATTCACTGTC CGAACCTGAA ATGGCAGCTC

2401 TTCGAGATTT TGTGGCAAGA AATGTAAAAG ATGGGCTAAT TACTCCAACG ATTGCACCTA

2461 ATGGAGCCCA AGTTCTCCAG GTGAAGAGGG GGTGGAAACT GCAAGTTTCT TATGATTGCC

2521 GAGCTCCAAA CAATTTTACT ATCCAGAATC AGTATCCTCG CCTATCTATT CCAAATTTAG

2581 AAGACCAAGC ACACCTGGCA ACGTACACTG AATTCGTACC TCAAATACCT GGATACCAAA

2641 CATACCCCAC ATATGCCGCG TACCCGACCT ACCCAGTAGG ATTCGCCTGG TACCCAGTGG

2701 GACGAGACGG ACAAGGAAGA TCACTATATG TACCTGTGAT GATCACTTGG AATCCACACT

2761 GGTACCGCCA GCCTCCGGTA CCACAGTACC CGCCGCCACA GCCGCCGCCT CCACCACCAC

2821 CACCGCCGCC GCCTCCATCT TACAGTACCC TGTAAATACC TGTCATGTCC TTCAGGATCT

2881 CTGCCCTCAA AATTTATTTCC TGTTCAGCTT CTCAATCAGT GACTGTGTGC TAAATTTTAG

2941 GCTACTGTAT CTTCAGGCCA CCTGAGGCAC ATCCTCTCTG AAACGGCTAT GGAAGGTTAG

3001 GGCCACTCTG GACTGGCACA CATCCTAAAG CACCAAAAGA CCTTCAACAT TTTCTGAGAG

3061 CAACAGAGTA TTTGCCAATA AATGATCTCT CATTTTTCCA CCTTGACTGC CAATCTAACT

3121 AAAATAATTA ATAAGTTTAC TTTCCAGCCA GTCCTGGAAG TCTGGGTTTT ACCTGCCAAA

3181 ACCTCCATCA CCATCTAAAT TATAGGCTGC CAAATTTGCT GTTTAACATT TACAGAGAAG

3241 CTGATACAAA CGCAGGAAAT GCTGATTTCT TTATGGAGGG GGAGACGAGG AGGAGGAGGA

3301 CATGACTTTT CTTGCGGTTT CGGTACCCTC TTTTTAAATC ACTGGAGGAC TGAGGCCTTA

3361 TTAAGGAAGC CAAAATTATC GGTGCAGTGT GGAAAGGCTT CCGTGATCCT CTCGCTGCAC

3421 CCTTAGAAAC TTCACCGTCT TCAAACTCCA TTTCCATGGT TCTGTTAATT CTCAAGGAGC

3481 AGCAACTCGA CTGGTTTCTCC CAGGAGCAGG AAAAACCCTT GTGACATGAA ACATCTCAGG

3541 CCTGAAAAGA AAGTGCTCTC TCAGATGGAC TCTTGCATGT TAAGACTATG TTCTTCACATC

3601 ATGGTGCAAA TCACATGTAC CCAATGACTC CGGCTTTGAC ACAACACCTT ACCATCATCA

3661 TGCCATGATG GCTTCCACAA AGCATTAAAC CTGGTAACCA GAGATTACTG GTGGCTCCAG

3721 CGTTGTTAGA TGTTCATGAA ATGTGACCAC CTCTCAATCA CCTTTGAGGG CTAAAGAGTA

3781 GCACATCAAA AGGACTCCAA AATCCCATAC CCAACTCTTA AGAGATTTGT CCTGGTACTT

3841 CAGAAAGAAT TTTCATGAGT GTTCTTAATT GGCTGGAAAA GCACCAGCTG ACGTTTTGGA

3901 AGAATCTATC CATGTGTCTG CCTCCATATG CATCTGGGCA TTTCATCTTC AGTCCCCTCA

3961 TTAGACTGTA GCATTAGGAT GTGTGGAGAG AGGAGAAATG ATTTAGCACC CAGATTCACA

4021 CTCCTATGCC TGGAAGGGGG ACATCTTTGA AGAAGAGGAA TTAGGGCTGT GGACACTGTC

4081 TTGAGGATGT GGACTTCCTT AGTGAGCTCC ACATTACTTG ATGGTAACCA CTTCAAAAAGG

4141 ATCAGAATCC ACGTAATGAA AAAGGTCCCCT CTAGAGGATG GAGCTGATGT GAAGCTGCCA

4201 ATGGATGAAA AGCCTCAGAA AGCAACTCAA AGGACTCAAA GCAACGGACA ACACAAGAGT

4261 TGTCTTCAGC CCAGTGACAC CTCTGATGTC CCCTGGAAGC TTTGTGCTAA CCTGGGACTG

4321 CCTGACTTCC TTTAGCCTGG TCCCTTGCTA CTACCTTGAA CTGTTTTTATC TAACCTCTCT

4381 TTTTCTGTTT AATTCTTTGC TACTGCCATT GACCCTGCTG CAGGATTTGT GTCATTTTCC

4441 TGCCTGGTTG CTGAGACTCC ATTTTGCTGC CACACACAGA GATGTAAGAG GCAGGCTTTA

4501 ATTGCCAAAG CACAGTTTGA GCAGTAGAAA ACAACATGGT GTATATCTCA AATTGCCTGA

4561 CATGAAGAGG AGTCTAACGG TGAAGTTTCA CTTTTCATCA GCATCATCTT TCACATGTTC

4621 ATTATCATCT GCTCTTATTC TTGCATGTTT AAACACTTAA AATTTTTAGT ATAATTTTTA

4681 GTGTGTTTTG AAGTGGTGAC TAGGCTTTCA AAAACTTCCA TTGAATTACA AAGCACTATC

4741 CAGTTCTTAT TGTTAAACTA AGTAAAAATG ATAAGTAACA TAGTGTAAAA TATTCCTTTA

4801 CTGTGAACTT CTTACAATGC TGTGAATGAG AGGCTCCTCA GAACTGGAGC ATTTGTATAA

4861 TAATTCATCC TGTTCATCTT CAATTTTAAC ATCATATATA ATTTCAATTC TATCAATTGG

4921 GCCTTTAAAA ATCATATAAA AGGATATAAA ATTTGAAAAG AGAAACCTAA TTGGCTATTT

4981 AATCCAAAAAC AACTTTTTTT TTCCTTCAAT GGAATCAGAA AGCTTGTCAA TCACTCATGT

5041 GTTTTAGAGT AATTACTTTTT AAAATGGTGC ATTTGTGCTT CTGAACTATT TTGAAGAGTC

5101 ACTTCTGTTT ACCTCAAGTA TCAATTCATC CTCCATACAT TTGAATTCAA GTTGTTTTTT

5161 TGTCAAATTT ACAGTTGTCA ATTGATCTTC AAGCTGCAGG GTGCCTAGAA ATGGGCCGTT

5221 GTCTGTAGCC CTGGCATGTG CACACGGACA TTTGCCACCA CTGCAAGCAA AAGTCTGGAG

5281 AAGTTCACCA ACGACAAGAA CGATTAGGGA AAATATGCTG CTGTGGGTTA ACAACTCAGA

5341 AAGTCCCTGA TCCACATTTG GCTGTTTACT AAAGCTTGTG ATTAACTTTT TGGCAGTGTG

5401 TACTATGCTC TATTGCTATA TATGCTATCT ATAAATGTAG ATGTTAAGGA TAAGTAATTC

5461 TAAATTTATT ATTCTATAGT TTTGAAGTTT GGTTAAGTTT CCTTTCACTC AATTGATTTA

5521 TTTTGTTGTT AATCAAATTT ATGTTAATTG GATCCTTTAA ATTTTTTTTG GCATTTTCCA

5581 ACAAAAATGG CTTTATTCAT AAGAAAGGAA AAAAATCAAT GGAATTTGAT ATCTAAAGAA

5641 GTTAGAAAGG GAGCAAAATA AAAAACATAA AGGAGATAGA TGAATTAGTA AGCAAATCAG

5701 TAGTCGAGTT TTTCAAACTG GCAAAATTAA TTAATTGACT TTTAGCCCAA ATTTACATTG

5761 TTAATTAAAT CAAGAAGGAA GAAGATCTAA GAGCTCCCAT TGATAGGCAA GCCTAGAGAG

5821 AACTAGCTAA ATTTATCATG CTAGGATATT GAAACACAGA AAGTTTACAT ACATTTATGA

5881 AGGGTCAATT TAGTTTGGAC AGTGAGGTAT TTGTCTTAGT GGAAAAAAGG AGAATTAGTC

5941 TGATCAAATC GTGAAGTAAT ACAGTGAACT TGCAGGTGCA CAAAATAAGA GGGCCACATC

6001 TATATGGTGC AGTCTGGAAT TCTGTTTAAG TTTGTAGGTA CCTCTTGGAC TTCTGAATTG

6061 ATCCAGTTGT CATCCACCAC AGACATCTCA CATCAGATAC AGTTCCAAGA TTGACAACAG

6121 AGAACAACCT GCTGGAAAGA CCTGGGCAGA AATGGAGAGC CCTGCGGGAA CCATGCTACA

6181 TTTTCATCTA AAGAGAGAAT GCACATCTGA TGAGACTGAA AGTTCTTTGT TGTTTTAGAT

6241 TGTAGAATGG TATTGAATTG GTCTGTGGAA AATTGCATTG CTTTTATTTC TTTGTGTAAT

6301 CAAGTTTAAG TAATAGGGGA TATATAATCA TAAGCATTTT AGGGTGGGAG GGACTATTAA

6361 GTAATTTTAA GTGGGTGGGG TTATTTAGAA TGTTAGAATA ATATTATGTA TTAGATATCG

6421 CTATAAGTGG ACATGCGTAC TTACTTGTAA CCCTTTACCC TATAATTGCT ATCCTTAAAG

6481 ATTTCAAATA AACTCGGAGG GAACTGCAGG GAGACCAACT TATTTAGAGC GAATTGGACA

6541 TGGATAAAAA CCCCAGTGGG AGAAAGTTCA AAGGTGATTA GATTAATAAT TTAATAGAGG

6601 ATGAGTGACC TCTGATAAAT TACTGCTAGA ATGAACTTGT CAATGATGGA TGGTAAATTT

6661 TCATGGAAGT TATAAAAGTG ATAAATAAAA ACCCTTGCTT TTACCCCTGT CAGTAGCCCT

6721 CCTCCTACCA CTGAACCCCA TTGCCCCTAC CCCTCCTTCT AACTTTATTG CTGTATTCTC

6781 TTCACTCTAT ATTTCCTCT ATTTGCTAAT ATTGCATTGC TGTTACAATA AAAATTCAAT

6841 AAAGATTTAG TGGTTAAGTG C

<210> 2

<211> 568

<212> PRT

<213> Homo sapiens

<400> 2

1 MCHKLTDGRF SGATIQNEEQ RYRSCRQKTE LRFTCAAKSE LQKSSTTKNA

51 TKTMRLASPT TALHCAARHM NCRAPPPTAP PQSRLPCSAV TQSPCATMRP

101 SQARAPSSRA RDFWIQDHRS EAPHELVGRG SALLVSGRHG DVRRAQHADQ

151 GSDGTLWCLF GSVGWYRAIN KRVLKTKKRR SGRGGQDPGL HPHRSEATAG

201 RSPPTPTVTL GPDCPPPPPP PPPNNNNNNNN SKHTGHKSAC VPNMTERRRD

251 ELSEEINNLR EKVMKQSEEN NNLQSQVQKL TEENTTLREQ VEPTPEDEDD

301 DIELRGAAAA AAPPPPIEEE CPEDLPEKFD GNPDMLAPFM AQCQIFMEKS

351 TRDFSVDRVR VCFVTSMMTG RAARWASAKL ERSHYLMHNY PAFMMEMKHV

401 FEDPQRREVA KRKIRRLRQG MGSVIDYSNA FQMIAQDLDW NEPALIDQYH

451 EGLSDHIQEE LSHLEVAKSL SALIGQCIHI ERRLARAAAA RKPRSPPRAL

501 VLPHIASHHQ VDPTEPVGGA RMRLTQEEKE RRRKLNLCLY CGTGGHYADN

551 CPAKASKSSP AGNSPAPL

Claims (10)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of human PEG protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 1-1707 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 1-1707 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.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 1-1707 position.

4. isolated human PEG protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 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.2 polypeptide of sequence.

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

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

8. the preparation method with polypeptide of human PEG protein active is characterized in that, comprises the steps:

(1) nucleotide sequence that coding is had a polypeptide of human PEG protein-active operationally is connected in expression regulation sequence, form the human PEG protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 1-1707 position among described nucleotide sequence and the SEQ ID NO.1;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of human PEG;

(3) under the condition that is fit to expressing human PEG10 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with human PEG protein-active.

9. energy and the described human PEG protein polypeptide of claim 4 specificity bonded antibody is characterized in that, comprise polyclonal antibody and monoclonal antibody.

10.PEG10 gene is as the application of diagnosing cancer of liver mark.