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

Abstract

The invention discloses a high expression liver cancer gene PEG10. The invention also discloses the encoding protein of gene PEG10; in addition, the invention also discloses the application of PEG10 gene as a diagnostic marker of liver cancer. Gene PEG10 of the invention is a high expression gene of liver cancer, the expression rate of PEG10 in liver cancer is up to 66%, but its expression rate and level in the liver tissue around the cancer is extremely low. Therefore PEG10 polynucleotide, PEG10 protein and its antibody, as well as the antagonist, agonists, etc related to PEG10 protein possess an enormous application prospect as they can provide novel therapeutic approach for the treatment of variety of illnesses including liver cancer.

Description

Liver cancer high-expression gene PEG 10, its proteins encoded and application thereof

Technical field

The present invention relates to biological technical field, particularly, the present invention relates to a kind of mainly the imprinted genes PEG10 and the proteins encoded thereof of high expression level in the human liver cancer tissue; In addition, the invention still further relates to the application of PEG10 gene.

Background technology

Famous biologist Nobel laureate RenatoDulbecco took the lead in proposing " Human Genome Project " (Human Genomic Pr mountain ect on the Science magazine in 1986, be called for short HGP), the proposition of this suggestion has caused scientific circles and has reached 3 years debating actively.United States Government's decision in October nineteen ninety provides funds 3,000,000,000 dollars formally to start " Human Genome Projects ", anticipates the full gene sequence (about 3,000,000,000 complete nucleotide sequences altogether) of taking human body in 2005; Study its interaction and gene function subsequently, thereby open human all mysteries of genetic information, make human understanding reach a new height self.

The Human Genome Project can be described as since the dawn of human civilization century one of the engineering of the greatest understanding self.In the end of the year 1997, the ultimate aim that the someone proposes HGP should provide the biological cycles table, and " element " of this periodictable is exactly ten thousand genes of 10-14 of human all proterties of decision.Finish the order-checking of 3,000,000,000 bases and only laid structural basis (being structural genomics), and the functional study (functional genomics) of comparative genomics (comprising its encoded protein matter) and whole genome could directly be brought into play its vital role on genomic value is found for this target.Development along with genomics, people design and have created many important biomolecules, be widely used in each field such as pharmacy, agricultural, food, chemical industry, makeup, environment, the energy, not only can promote the progress of science, also will produce surprising economic benefit, thereby the initiation Industrial Revolution forms based on the life science industry of genomics.Human limited genetic resources WKG working disposable distribution, obtains the most effective and maximum enterprise of quantity of gene, is expected to utilize its gene patent to monopolize following biological and pharmaceutical industry market.Bill. Gates once said: the people that the next one creates bigger wealth will appear at the gene field.

Along with the fast development of genome plan, capture the malignant tumour that comprises liver cancer and will become a reality in the near future.China is primary hepatocyte hepatocarcinoma (primaryhepatocellularcarcinoma, HCC) hotspot, its mortality ratio has accounted for first and second position in China part rural area and city, it is the major disease that has a strong impact on China's people ' s health, have every year 38.6 ten thousand people to die from liver cancer in the world, and wherein 45% be in China.Think at present, the generation of liver cancer, development, also the same with other malignant tumour, it is the complex process of polygene, multifactor participation, comprise the losing of somatic cell gene sudden change, cancer suppressor gene, oncogene active and cross and express or the like, many oncogenes, cancer suppressor gene, the abnormal activation or the inactivation that reach genes involved are the molecular basises that liver cancer takes place.Theoretically, should there be more gene to participate, but many genes of having found at present, its detailed function is still not fully aware of.At present proved that oncogene relevant with human liver cancer or unconventionality expression gene comprise; PTEN, p21, p27, p73, p15, p53, RBl, APC, nm23, P16, MXR7, IGF-I, TGFO, HGF-R (C-met)/HGF, c-fins (CSF-IR), c-erbB-1 (EGF-R)/c-erbB-2 (neu), Ras, Raf, c-myc, c-ets-2 or the like, but none is the liver cancer-specific expressing gene, therefore, seek new liver cancer unconventionality expression or missing gene, be familiar with hepatocellular growth from gene level, differentiation, the molecule mechanism of regeneration and canceration, and illustrate its signal transduction process and approach, important biological significance is not only arranged, and for diagnosis of the early gene of liver cancer and information therapy, all have great clinical application meaning and economic development value, in the research of human genome, also can capture new commanding elevation.

Fast development along with Protocols in Molecular Biology, the novel method of searching, isolated genes constantly occurs, mainly contain following four kinds: 1, from the method for eDNA or mRNA: utilizing difference Song of cellular gene expression to separate Disease-causing gene is a class important method, makes fast progress at present.Comprise cDNA subtractive hybridization method, mRNA differential display technique, EST (Expressed sequence tags) differential hybridization screening etc.; 2, start with from analysing protein; According to proteic position, set out in conjunction with characteristics or partial function, separate desirable proteins earlier, obtain corresponding gene again.Comprise immuno-precipitation, two-dimensional electrophoresis method, yeast two-hybrid system method etc.; 3, from seeking genomic DNA fragment: search out and closely linked genetic marker in target gene seat or partial function information, thereby determine the position of gene certain section in genome that certain proterties is relevant, and then utilize different methods to find the intrachromosomal expressed sequence of this section, finally be cloned into goal gene.Comprise homologous sequence hybrid method, PCR direct election method, exon trapping, concensus sequence cloning etc. between complicated probe sieve storehouse method, Northern hybrid method, splice site sieve method, CpG island sieve method, kind; 4, from full genome: the clone who directly separates this gene according to the effect of related gene, and needn't find out its biochemical function or collection of illustrative plates location in advance, do not need number or its interaction mode of hypothetical gene yet, comprise genome mismatch scanning and representational difference analysis etc.The method of the new gene of above-mentioned searching cuts both ways, and uses any method, should maximize favourable factors and minimize unfavourable ones according to breadboard condition and advantage separately, takes practicable method.

First Application mRNA difference technique of display such as American scholar LiangPeng were separated and evaluation different tissues and intercellular genetic expression in 1992, use this method, it has been found that a large amount of unknown genes, as Liang etc., the mRNA that compares normal breast and mammary cancer with this method, found the S1 gene of total length 600bp: (ChenSL such as Chen, MaroulakouIG, GreenJE, et al.Isolation and characterizationofnovelgene expressed inmultiple cancers, Oncogene, 1996:12 (4): 741-751) found a new gene N8 with this method, total length 2.4kb is positioned on the 8q13 karyomit(e), crosses in cancerous lung tissue and expresses; (ShibaharaK such as Japan scholar Shibabara; AsanoM; IshidaY; Aoki T; KoikeT; Ho mountain OT, IsolationofanovelmousegeneMA-3thatiSinduceduponprogramme d celldeath, Gene, 1995Dec, 166 (2): 297-301) be separated to a new gene M A-3 who causes programmed cell death with this method, or the like.But the DDPCR technology remains at present in some shortcomings, though some scholars have done many improvement in this respect in recent years, as the reasonableness of design of primers, how to reduce false positive etc., but still need constantly perfect, believe the constantly perfect of DDPCR technology, will in life science, become very useful instrument.

Therefore, to research and develop in liver cancer the gene and/or the albumen of high expression level significant for treatment and diagnostic purpose.This area press for new in liver cancer the gene and/or the albumen of high expression level.

Imprinted genes PEG10 is positioned on the human chromosomal 7q21, and PEG10 is positioned near the SGCE gene, and its mouse homolog shows recently by trace.Therefore in human chromosomal 7q21, there is the new imprinted genes group of a class probably.The analysis of the open reading frame of two kinds of precognitions (ORF1 and ORF2) shows that ORF1 and ORF2 are homologous with respect to some vertebrate protein.Placental PEG10 in early days the anoxic stage be low the adjusting, be highly active in week at the 11-12 of pregnancy.The external cause of PEG10 is expressed has effect to oncogenic activity.PEG10 protein is associated with media SIAH1, and the overexpression of PEG10 has reduced the necrocytosis of media SIAH1.Structure shows, imprinted genes PEG10 is at the liver regeneration of human hepatocytes and press down in the cancer and can play important effect.

In view of the critical function of imprinted genes PEG10, PEG10 is significant for the research and development imprinted genes.

Summary of the invention

One of the technical problem to be solved in the present invention provide a kind of in liver cancer the imprinted genes PEG10 (Paternally Expressed 10) of high expression level.

Two of the technical problem to be solved in the present invention provides a kind of people's albumen PEG10.

Three of the technical problem to be solved in the present invention provides the application of PEG10 gene as the diagnosing cancer of liver mark.

The present invention obtains a gene fragment from liver cancer tissue, and then has cloned one and the new full length gene cDNA of PEG10 positive correlation sequence, this gene order and primary hepatocyte hepatocarcinoma height correlation by gene clone technology.The expression of this gene in liver cancer that interesting is is up to 66%, and the other hepatic tissue of cancer is not expressed or express extremely low.One 568 amino acid whose protein of this genes encoding are positioned cytoplasm.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of human PEG protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 1-1707 position dna molecular 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.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 1-1707 position.

In another aspect of this invention, provide a kind of isolated human PEG protein and peptide, 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.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.

In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.

In another aspect of this invention, also provide a kind of preparation method with polypeptide of human PEG protein active, this method comprises:

(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.

Preferably, the nucleotide sequence that uses in the method has the sequence of 1-1707 position among the SEQ ID NO.1.

The present invention also provides and PEG10 protein polypeptide specificity bonded antibody.

In the present invention, " isolating " DNA is meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with the protein of following it in cell.

In the present invention, term " human PEG albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with human PEG protein-active is as 1-1707 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 1-1707 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 1-1707 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 1-1707 position.Wherein, " stringent condition " is meant that nucleotides sequence is listed in the film condition of washing after the hybridization on the film.For example, in the art, low stringency is washed film can pour 150ml left and right sides washing lotion in hybrid pipe, put into Hybond membrane, room temperature continued to shake about 20 minutes, and high tight degree to wash film can be to pour 200ml left and right sides washing lotion in hybrid pipe into, put into Hybond membrane, continue to shake about 20 minutes in 50 ℃ of shaking tables.This term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 1-1707 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.1 with the human PEG identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, term " human PEG albumen or polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of PEG10 protein-active.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural human PEG identical function.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of PEG10 and reactive derivative.

The variant form of inventor PEG10 polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of human PEG DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-human PEG polypeptide to obtain.The present invention also provides other polypeptide, as comprises human PEG polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of human PEG polypeptide.Usually, this fragment have the human PEG peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

In the present invention, " human PEG conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.2, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			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；Ala	Leu

Invention also comprises the analogue of human PEG albumen or polypeptide.The difference of these analogues and natural human PEG polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing human PEG polypeptide of the present invention, the PEG10 encoding sequence operationally can be connected in expression regulation sequence, thereby form the human PEG protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

The present invention also provides the antibody special to human PEG, comprises polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare special antibody at PEG10.For example, the human PEG gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human PEG10 or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can be with each (for example, Kohler et al., Nature 256:495,1975 of hybridoma technology system; Kohler et al., Eur.J.Immunol.6:511,1976; Kohler et al., Eur.J.Immunol.6:292,1976).Antibody of the present invention comprises the antibody that can prevent the human PEG function, also can be the antibody that does not influence the human PEG function.Each antibody-like can produce by the fragment of human PEG gene product or functional domain are caused immunity, and human PEG gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the PEG10 gene product bonded antibody of non-modified forms, can come immune animal to obtain by being used in the gene product that prokaryotic cell prokaryocyte for example produces among the E.coli.With posttranslational modification form such as glycosylation or phosphorylated protein or polypeptide bonded antibody, can obtain by the immune animal that comes that is used in the gene product that produces in eukaryotic cell such as yeast or the insect cell.

People PE610 antibody of the present invention can be used for identifying the cell of expressing human PEG10 albumen or polypeptide, as the JurkatT cell.For example, can with a kind of detectable molecule for example fluorescein isothiocyanic acid (FITC) come mark PEG10 specific antibody, allow the human PEG specific antibody contact then, detect and PEG10 specific antibody bonded cell with fluorescent microscope or flow cytometer again with cell sample.

Except cell surface detects human PEG, can also analyze this protein with the Western engram technology.Cell pyrolysis liquid can from culturing cell or take from patient's tissue sample such as suprarenal gland extract, and be dissolved in the lysis buffer that contains stain remover.Use sds polyacrylamide gel electrophoresis isolated cell extract (simultaneously with the human PEG polypeptide of purifying as positive control) then, then it is transferred on the nitrocellulose by electrophoresis hybridization.In order to survey the PEG10 polypeptide, can use typical antibodies detection method, for example radioautograph or alkaline phosphatase enzyme assay method with the immunity of Western trace.And can use the contrast of immunization serum or incoherent monoclonal antibody as non-specific responding.

Whether and quantity the expression of also available Nothern blotting technical Analysis PEG10 gene product, the i.e. existence of rna transcription thing in cell of analyst PEG10.

The Nothern engram analysis of human PEG DNA and the Western engram analysis of human PEG specific antibody can be united use, with the expression of confirmer PEG10 in biological specimen.Human PEG DNA can also be used for Southern engram analysis or in situ hybridization analysis, with this assignment of genes gene mapping on karyomit(e), and can carry out genetic linkage analysis to find out other possible disease related gene.

Human PEG Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book contriver PEG10.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

Utilize human PEG albumen of the present invention, by various conventional screening methods, can filter out with human PEG take place interactional material or, as acceptor, inhibitor or antagonist etc.

Inventor PEG10 albumen and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

The present invention also provides the application of PEG10 gene as the diagnosing cancer of liver mark.PEG10 gene of the present invention is a kind of liver cance high-expression gene, and the expression rate of PEG10 in liver cancer is up to 66%, and the expression rate and the expression level of hepatic tissue are extremely low by cancer.Therefore, the PEG10 gene can be used as the diagnosing cancer of liver mark, make to its in depth study might quantification liver cancer the detection index, for further clinical expansion is contributed.In addition, PEG10 polynucleotide, PEG10 albumen and antibody thereof, and the relevant antagonist of PEG10 albumen, agonist etc. can be treatment and comprise that multiple disease such as liver cancer provides new treatment approach, thereby have great application prospect.

Description of drawings

Fig. 1 is that N is a cancer beside organism by the expression figure of RT-PCR checking imprinted genes PEG10 of the present invention in liver cancer/cancer beside organism, and C is a cancerous tissue, and β-actin is as internal reference.

Embodiment

The present invention is further detailed explanation below in conjunction with drawings and Examples.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

1. separate tissue (Tissue isolation)

Derive from the operation patients (cancer is other negative in that liver cancer is all positive for RT-PCR confirmation AFP) that HBV is positive and express the primary hepatocarcinoma of alpha-fetoprotein.The liver of excision cuts focus rapidly and reaches 5 centimeters outer cancer beside organisms on every side once exsomatizing, and puts into liquid nitrogen (80 ℃) and preserves.The other diagnosis of cancer and cancer is final foundation with pathological diagnosis all.

2. the extraction agent box of total RNA

Extracting RNA reagent employing TRIzol reagent (GIBCO/BRL), this reagent are based on the extraction process production of one step of acidic phenol.Being used for used vessel of extracting RNA and water all will not have the processing of RNA enzyme, to guarantee the environment of no RNA enzyme in the experiment.

3.RNA extraction steps

To grind vessel such as pestle and homogenizer and do roasting 4h, remove the RNA enzyme, cooling at 200 ℃; Add precooling in the liquid nitrogen, will organize from liquid nitrogen and take out rapidly, be crushed into powder; With curet tissue is put into the homogenizer that adds TRIzol reagent in advance, homogenate number minute; Liquid after the homogenate is changed in the centrifuge tube of no RNA enzyme, behind the adding chloroform, 4 ℃ of centrifugal layerings; The upper strata water is changed in the centrifuge tube of a no RNA enzyme, add Virahol, 4 ℃ of centrifugation RNA; Precipitate 2 times with 75% washing with alcohol; Deionized water dissolving precipitation with no RNA enzyme.Extractive RNA quality evalution: ultraviolet spectrophotometer is measured 260/280 ratio (ratio is all 1.7～2.0); And observation has or not degraded in MOPS denaturing formaldehyde glue.

4.cDNA synthetic

Get total RNA2 μ g, OligodT ₁₆1 μ L, 70 ℃ of insulation 3min, sex change 5min on ice immediately.Add 5 * buffer, the reversed transcriptive enzyme of each 2 μ L of the dNTP of DTT and 50mg/L and 1 μ L, behind the abundant mixing, 42 ℃ of 2h.Template uses final concentration to be generally 1 μ g/100 μ L.

5.RT-PCR amplification

The design primer, PEG10: the forward direction primer is 5 '-TGCTTCTGGCAACTTCATTG-3 ', the back is 5 '-TCAAATGACAGCACCTCTCG-3 ' to primer; Beta-actin: the forward direction primer is 5 '-TCACCCACACTGTGCCCATCTACGA-3 ', and the back is 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 ' to primer.Make internal reference with β-actin, each composition is in the reaction mixture: β-actin (F), β-actin (R), PEG10 (F), PEG10 (R), 10 * Buffer, MgCl ₂, dNTP, Taq archaeal dna polymerase, cDNA template be respectively 0.2,0.2,0.4,0.4,1.0,1.0,0.2,0.1 and 5 μ LcDNA templates, replenishes ddH at last ₂It is 10 μ L that O makes reaction system.The reaction conditions of PCR is 94 ℃ of sex change 5min; 94 ℃ of 30s of each circulation, 53 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations then; Last 72 ℃ are extended 7min.

6. result

The result is as shown in Figure 1: utilize the RT-PCR technology to detect PEG10 expression of gene difference in 32 pairs of liver cancer and cancer beside organism, finding has 24 routine PEG10 genes obviously to raise in 32 examples, and promptly the expression rate of PEG10 gene in liver cancer tissue is up to 66%.

Embodiment 2

The preparation of human PEG polypeptide and purification

In this embodiment, the PEG10 encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.

The human PEG polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.

1. construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.1) according to human PEG, design amplifies complete coding and reads the primer of frame (corresponding respectively to about 20 above Nucleotide of encoding sequence 5 ' and 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pGEX-2T carrier of selecting for use) respectively, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with the human PEG gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl ₂Method changes bacillus coli DH 5 alpha over to, and Screening and Identification obtains containing the engineering bacteria DH5 α-pGEX-2T-PEG10 of pGEX-2T-PEG10 expression vector.

2. express the isolation identification of the engineering bacteria of GST-PEG10 recombinant protein

DH5 α-pGEX-2T-PEG10 the engineering bacteria of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-PEG10 fusion rotein.

3.GST-PEG10 the extraction purifying of fusion rotein

The proteic engineering bacteria DH5 of abduction delivering GST-PEG10 amalgamation and expression α-pGEX-2T-PEG10 as stated above.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines GST-PEG10 in centrifugal 10 minutes, abandoned supernatant.Clean twice by the amount that every milliliter of ultrasonic liquid gained precipitation adds 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 31kDa place is human PEG albumen.

Sequence table

＜110〉Research Center of Shanghai Human Genome

＜120〉liver cancer high-expression gene PEG 10, its proteins encoded and application thereof

<130> NP-10015

<160> 2

<210> 1

<211> 6861

<212> DNA

＜213〉homo sapiens (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 GGAAAAGAGC 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 AATTTATTCC 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 CTGGTTCTCC CAGGAGCAGG AAAAACCCTT GTGACATGAA ACATCTCAGG

3541 CCTGAAAAGA AAGTGCTCTC TCAGATGGAC TCTTGCATGT TAAGACTATG TCTTCACATC

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 CTTCAAAAGG

4141 ATCAGAATCC ACGTAATGAA AAAGGTCCCT CTAGAGGATG GAGCTGATGT GAAGCTGCCA

4201 ATGGATGAAA AGCCTCAGAA AGCAACTCAA AGGACTCAAA GCAACGGACA ACACAAGAGT

4261 TGTCTTCAGC CCAGTGACAC CTCTGATGTC CCCTGGAAGC TTTGTGCTAA CCTGGGACTG

4321 CCTGACTTCC TTTAGCCTGG TCCCTTGCTA CTACCTTGAA CTGTTTTATC 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 AATCCAAAAC AACTTTTTTT TTCCTTCAAT GGAATCAGAA AGCTTGTCAA TCACTCATGT

5041 GTTTTAGAGT AATTACTTTT 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 ATTTCTCTCT ATTTGCTAAT ATTGCATTGC TGTTACAATA AAAATTCAAT

6841 AAAGATTTAG TGGTTAAGTG C

<210> 2

<211> 568

<212> PRT

＜213〉homo sapiens (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 PPPNNNNNNN 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.