CN1629637A - Tumor relevant secretory protein as a liver cancer marker and uses thereof - Google Patents

Abstract

This invention provides MAGEDI protein-a new carcinoma of liver marker and the usage of MAGEDI protein and its code sequence, such as diagnosis of carcinoma of liver. This invention also provides kits for testing it.

Description

As relevant secretory protein of the tumour of liver cancer marker and uses thereof

Technical field

The invention belongs to biotechnology and medical domain, specifically, the present invention relates to the relevant secretory protein 1 of a kind of new liver cancer marker-tumour (Melanoma antigen gene, family D 1, abbreviate MAGED1 albumen as).The invention also discloses the purposes of MAGED1 albumen and coded sequence thereof, as be used for diagnosing tumour, and the pharmaceutical composition that contains MAGED1 protein antagonist (as antibody and antisensenucleic acids).

Background technology

Twentieth century since the fifties medical science development of leap has been arranged, the progress of treatment of cancer also is fruitful, and found many directly with relevant oncogene and the tumor suppressor gene of cancer generation.

Up to now, in melanoma and comprise in the various tumor tissues of prostate cancer, thymic carcinoma, oophoroma, stomach cancer and found many tumour antigens.The human tumor immunological rejection antigen of having found at present can be divided into four types.First kind antigen comes from the somatic mutation of normal gene product, and the second class antigen comes from the gene mutation relevant with oncogenic process.This two classes antigen is patient-specific antigen, is not suitable for the ubiquity treatment.The 3rd class antigen is the sort of expression to be arranged also in normal structure, but the product of the gene that expression raises in tumour.If do not relate to sudden change, this class antigen has ubiquity in various cancer patients, but they are normally tissue-specific, and is not peculiar by tumour, has little significance in clinical practice.The 4th class antigen has strict tumour-specific, and is relevant with general oncogenic process.Therefore, this class antigen is expressed in human tumor widely, is suitable for the target spot as tumor markers and antineoplastic immune attack most.In addition, the tumor markers that is particularly suitable for diagnosing is the albumen of those secretion property.Yet, seldom belong to this class in the antigen of having found at present.

The MAGED1 gene is a member of MAGE family.Its accession number is AF258554, and nucleotide sequence is shown in SEQ IDNO:1, and amino acid sequence is shown in SEQ ID NO:2.Different with other MAGE family gene, the MAGED1 gene is all expressed in normal adult tissue, lacks tumour-specific (Genomics 1999 Jul15; 59 (2): 161-7).

Document (Neuro 2000 Aug are arranged; 27 (2): 279-88) report, the MAGED1 gene is relevant with the apoptosis of neurocyte.But this gene any report relevant not before the present invention with liver cancer genesis and development.

Be of great significance in order to treat with the relevant secretory protein of diagnostic purpose exploitation tumour.Therefore, this area presses for the tumor markers of the new secretion of exploitation, is used for the diagnosis and the treatment of tumour.

Summary of the invention

The purpose of this invention is to provide a kind of new people's liver cancer marker MAGED1 albumen with and fragment, analog and derivant.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and coded sequence.

The present invention finds first that through extensive and deep research MAGED1 can be used as liver cancer marker.This expression of gene product can detect in the serum of a lot of hepatocarcinoma patients, but the serum in normal person and other non-liver cancers patient lack detect less than.This shows, MAGED1 is the liver cancer marker of a special secretion.Finished the present invention on this basis.

In a first aspect of the present invention, the kit of a kind of detection tumour (especially liver cancer) is provided, it contains it and contains antibody and the instructions that combines with the MAGED1 protein-specific.Preferably, described MAGED1 contains the amino acid sequence of SEQ ID NO:2.

In another preference, the monoclonal antibody of described antibody.

In another preference, the polyclonal antibody of described antibody.

In another preference, described kit also contains positive control and negative control.

In another preference, described kit also contains sampling apparatus, as the device of blood sample collection.

In a second aspect of the present invention, a kind of protein-chip that detects cancer is provided, it comprises substrate and the point sample specific antibody in on-chip cancer associated protein, and wherein, the specific antibody of described cancer associated protein comprises the specific antibody of anti-MAGED1.

In another preference, the specific antibody of described cancer associated protein also comprises the antibody of the hepatoma associated protein that other are known, as resisting the antibody of following antigen: pTEN, p21, p27, p73, p15, p53, RB1, APC, nm23, P16, MXR7, IGF-II, TGF α, HGF-R, c-erbB-1, Ras, Raf, c-myc, c-ets-2.

In a third aspect of the present invention, the method that whether has MAGED1 albumen in a kind of test sample is provided, it comprises step:

The antibody of sample with the combination of MAGED1 protein-specific is contacted, observes whether form antibody complex, formed antibody complex and just represented to exist in the sample MAGED1 albumen,

Wherein, described sample is a blood sample.

In another preference, described blood sample is people's a blood sample.

In a fourth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the MAGED1 antagonist and the pharmaceutically acceptable carrier of safe and effective amount.Preferred L AGED1 antagonist is antibody and the MAGED1 antisense sequences of anti-MAGED1.These pharmaceutical compositions can be treated tumour.

In a fifth aspect of the present invention, provide the antibody that combines with people MAGED1 protein-specific.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people MAGED1 protein active is provided, and the compound that suppresses the expression of people MAGED1 albumen.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is the coded sequence of people MAGED1 albumen or the antisense sequences of its fragment.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and coded sequence.Polypeptide for example of the present invention can be used to screen the activator that promotes people MAGED1 protein active, perhaps suppresses the antagonist of people MAGED1 protein active or be used to the peptide finger-print to identify.The coded sequence of people MAGED1 albumen of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization reaction as probe, perhaps are used to make genetic chip or microarray.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1 has shown the expression of MAGED1 albumen.

Wherein, figure A is the expression before and after inducing, swimming lane 1. standard protein molecular weight; 2.pT ₇After 470-MAGED1-HMS174 (DE3) induces; 2.pT ₇Before 470-MAGED1-HMS174 (DE3) induces.

Figure B is a Western trace testing result (1: 10000), swimming lane 1.MAGED1 protein sample; 2.pT ₇470-MAGED1-HMS174 (DE3) induces preceding thalline.

Fig. 2 has shown the Western Blot result to the part patients serum.Each swimming lane is as follows: N: the normal person; K: hepatocarcinoma patient; Greatly: the hepatitis great three positive; Little: the hepatitis "small three positive".

Fig. 3 has shown carrier pT ₇470 structural drawing.

Detailed Description Of The Invention

In the present invention, " albumen of the present invention ", " polypeptide of the present invention " refer to Melanoma antigen gene family D1 or its active tablet Section and derivative.

In the present invention, " gene of the present invention ", " polynucleotides of the present invention " refer to encode Melanoma antigen gene family D1 or its The nucleotide sequence of active fragment and derivative comprises justice and antisensenucleic acids.

In the present invention, term " Melanoma antigen gene family D1 ", " Melanoma antigen gene family D1 " or " liver cancer marker MAGED1 " Be used interchangeably, all refer to have the albumen of people's liver cancer marker MAGED1 amino acid sequence (SEQ ID NO:2) or many Peptide.

As used herein, " separation " refers to that material separates (if natural thing from its primal environment Matter, primal environment namely are natural surroundingses). Not have such as the polynucleotides under the native state in the active somatic cell and polypeptide Separation and purification is arranged, but same polynucleotides or polypeptide as from native state with dividing in other materials that exist Open, then for separation and purification.

As used herein, it is natural that " Melanoma antigen gene family D1 of separation or polypeptide " refers to that Melanoma antigen gene family D1 is substantially free of Relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the egg of standard White matter purification technique purifying Melanoma antigen gene family D1. Basically pure polypeptide can produce on non-reduced polyacrylamide gel Give birth to single master tape.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. This Bright polypeptide can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique from protokaryon Or produce in the eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to The host that the recombinant production scheme is used, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analog of people's Melanoma antigen gene family D1. As used herein, term " sheet Section ", " derivative " refer to basically keep natural human Melanoma antigen gene family D1 of the present invention identical with " analog " Biological function or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be that (i) has one Individual or a plurality of conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and The amino acid residue of such replacement can be also can be by the genetic code coding, or (ii) one or The polypeptide that has substituted radical in a plurality of amino acid residues, or (iii) mature polypeptide and another compound (such as Prolong the compound of polypeptide half-life, for example polyethylene glycol) merge formed polypeptide, or (iv) additional amino Acid sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for purifying these are many The sequence of peptide or proteinogen sequence, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, These fragments, derivative and analog belong to the known scope of those skilled in the art.

In the present invention, term " people's Melanoma antigen gene family D1 " refers to have the SEQ ID NO of people's Melanoma antigen gene family D1 activity: The full-length polypeptide of 2 sequences or mature polypeptide. This term also comprises having with people's Melanoma antigen gene family D1 identical function (as short Advance the function of liver cancer cell growth), the variant form of SEQ ID NO.2 sequence. These variant forms comprise (but Be not limited to): one or more (be generally 1-50, preferably 1-30, more preferably 1-20, best 1-10) amino acid whose disappearance, insertion and/or replacement, and add one or several in that C end and/or N are terminal (being generally in 20, preferably is in 10, more preferably is in 5) amino acid. For example, in ability In the territory, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again Such as, in C end and/or one of the terminal interpolation of N or the common function that also can not change protein of several amino acid. This term also comprises active fragment and the reactive derivative of people's Melanoma antigen gene family D1.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, can be coded with the DNA of people MAGED1 DNA hybridization under high or low stringency condition Albumen and polypeptide or the albumen that utilizes the antiserum of anti-people's Melanoma antigen gene family D1 to obtain. The present invention also provides Other polypeptide, as comprise the fusion of people's Melanoma antigen gene family D1 or its fragment. Except the polypeptide of total length almost, The present invention has also comprised the soluble fragments of people's Melanoma antigen gene family D1. Usually, this fragment has people's Melanoma antigen gene family D1 order Row at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 companies Continuous amino acid is more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analog of people's Melanoma antigen gene family D1 or polypeptide. These analogs and natural human Melanoma antigen gene family D1 Difference can be difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, Perhaps have both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can pass through each Kind of technology obtains, and as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through the direct mutagenesis method Or the biological technology of other known moleculars. Analog also comprise have be different from the amino acid whose residue of natural L-(as D-amino acid) analog, and have that non-natural exists or synthetic amino acid analogue. Should be understood that this The polypeptide of invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(usually the not changing primary structure) form of modifying comprises: in the body or the chemically derived form of external polypeptide as Acetylation or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add Carry out glycosylation modified and polypeptide that produce during work step is rapid. This modification can be carried out sugar by polypeptide is exposed to The enzyme of baseization (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorus The sequence of acidifying amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Also comprise and being repaiied Thereby decorations have improved its anti-proteolysis performance or have optimized the polypeptide of solubility property.

In the present invention, " people's Melanoma antigen gene family D1 conservative variation polypeptide " refers to the amino acid with SEQ ID NO:2 Sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid at the most best Replaced by similar performance or close amino acid and form polypeptide.

Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, genomic DNA Or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding strand or noncoding strand. The coding region sequence of encoding mature polypeptide can with the identical or degeneracy of coding region sequence shown in the SEQ ID NO:1 Variant. As used herein, " variant of degeneracy " refers to that in the present invention coding has SEQ ID NO:2 Protein, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotides of the mature polypeptide of coding MAGED1 comprise: the coded sequence of an encoding mature polypeptide; Ripe The coded sequence of polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional code order Be listed as) and non-coding sequence. Term " polynucleotides of coded polypeptide " can be the multinuclear that comprises this polypeptide of encoding Thuja acid also can be the polynucleotides that also comprise additional code and/or non-coding sequence.

The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation The variant that allelic variant or non-natural take place. These nucleotide diversity bodies comprise that replacement variant, disappearance become Allosome and insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, It may be replacement, disappearance or the insertion of one or more nucleotides, but can be not many from changing in fact its coding The function of peptide.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization, comprise the nucleic acid fragment of justice and antisense. As used herein, the length of " nucleic acid fragment " contains 15 nucleotides at least, better is at least 30 nucleotides, Be more preferably at least 50 nucleotides, preferably more than at least 100 nucleotides. Nucleic acid fragment can be used for the expansion of nucleic acid Increase technology (such as PCR) to determine and/or to separate the polynucleotides of coding Melanoma antigen gene family D1.

People MAGED1 nucleotides full length sequence of the present invention or its fragment usually can use pcr amplification method, recombination method or Artificial synthetic method obtains. For the pcr amplification method, can be according to published relevant nucleotide sequence, especially Open reading frame sequence designs primer, and with commercially available cDNA storehouse or by routine well known by persons skilled in the art The prepared cDNA storehouse of method is as template, amplification and must relevant sequence. When sequence is longer, usually need into Twice of row 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 in large quantity relevant sequence with recombination method. This is common Be that it is cloned into carrier, change again cell over to, separate from the host cell after the propagation by conventional method then Obtain relevant sequence.

In addition, also available artificial synthetic method is synthesized relevant sequence, especially fragment length more in short-term. Logical Often, by first synthetic a plurality of small fragments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain code book invention albumen (or its fragment, derivative) by chemical synthesis Dna sequence dna. This dna sequence dna can be introduced then various existing dna molecular as known in the art (or as carries Body) and in the cell. In addition, also can will suddenly change by chemical synthesis and introduce in the protein sequence of the present invention.

The method of using round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available conventional method is synthetic.Available conventional method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetic engineering with carrier of the present invention or MAGED1 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technique.

By the recombinant DNA technology of routine, can utilize polynucleotide sequence of the present invention to can be used to express or produce the MAGED1 albumen of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or variant) of coding of the present invention people MAGED1 albumen, or with the recombinant expression carrier that contains these polynucleotide proper host cell that transforms or transduce;

(2). the host cell of in proper culture medium, cultivating;

(3). separation, protein purification from nutrient culture media or cell.

Among the present invention, people MAGED1 polynucleotide sequence can be inserted in the recombinant expression carrier.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain origin of replication, promoter, marker gene and translation control element usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people MAGED1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technique of body etc.Described dna sequence dna can effectively be connected on the suitable promoter in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate dihyrofolate reductase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic, or be used for colibacillary tetracycline or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promoter or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic, as bacterial cell; Or eukaryotic such as low, as yeast cells; Or higher eucaryotic cells, as mammalian cell.Representative example has: Escherichia coli, the bacterial cell of streptomyces; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or 293 cells etc.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotes such as Escherichia coli, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eucaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with conventional method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used nutrient culture media can be selected from various conventional nutrient culture media in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promoter of selection with suitable method (as temperature transition or chemical induction), cell is cultivated a period of time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cell membrane.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people MAGED1 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the antibody, polypeptide or other material that are used to screen antagonism MAGED1 protein function.

On the other hand, the present invention also comprises people MAGED1 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people MAGED1 gene outcome or fragment.Preferably, refer to that those can combine with people MAGED1 gene outcome or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.

The present invention not only comprises complete monoclonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.

The antibody of anti-people MAGED1 albumen can be used in the immunohistochemistry technology, detects the people MAGED1 albumen in the biopsy specimen.

Utilize albumen of the present invention,, can filter out with MAGED1 albumen interactional material takes place, as acceptor, inhibitor, activator or antagonist etc. by various conventional screening techniques.

Albumen of the present invention and antibody thereof, inhibitor, activator, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, but these materials are formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, 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): in intramuscular, the peritonaeum, intravenous, subcutaneous, intracutaneous or topical.

The present invention also provides a kind of pharmaceutical composition, and it contains MAGED1 antagonist of the present invention (as antibody and antisense sequences) and the pharmaceutically acceptable carrier or the excipient of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component be the treatment effective dose, for example every day about 1 microgram-10 mg/kg body weight.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people MAGED1 protein level.These tests are known in the art.The people MAGED1 protein level that is detected in the test can be used for diagnosing tumour.

The method that whether has MAGED1 albumen in a kind of test sample is to utilize the specific antibody of MAGED1 albumen to detect, and it comprises: sample is contacted with the MAGED1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MAGED1 albumen.

MAGED1 albumen or its polynucleotide can be used for the diagnosis and the treatment of MAGED1 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray or DNA chip, is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.The antibody of anti-MAGED1 can be fixed on the protein-chip, is used for the MAGED1 albumen of test sample.

The present invention also provides a kind of kit that detects liver cancer, the primer that it contains specific amplification MAGED1 to and/or the MAGED1 specific antibody.

In addition, in view of MAGED1 is the distinctive immunity antigen of tumour cell, be secreted in the body fluid.Therefore, the direct mensuration of the MAGED1 in blood sample or the urine also can be used as the foundation of early diagnosis of tumor except the observation index of the auxiliary diagnosis that can be used as tumour and prognosis.

Major advantage of the present invention is:

(1) MAGED1 exists only in the hepatocarcinoma patient serum, and can not detect in normal person or non-liver cancer patient's serum.Therefore, false positive rate is low.

(2) MAGED1 is a secreted protein, is convenient to be applied to serodiagnosis, the prognosis monitoring of liver cancer, and can be used as the target molecule of gene therapy, development genetically engineered drug or conduct design PTS.

Below in conjunction with specific embodiment, further set forth the present invention.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 people such as normal condition such as Sambrook, condition described in the molecular cloning laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1

The acquisition of the cDNA of MAGED1

Get human placenta, (GIBCO BRL company) extracts total RNA by manufacturer's instructions with Trizol reagent, extracts mRNA with the mRNA kit (Pharmacia company) of purifying.Utilize primer linker-primer (Stratgene): 5 '-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3 ' (SEQ ID NO:3), with MMLV-RT-Superscript II (GIBCO BRL company), reverse transcriptase is carried out reverse transcription reaction at 42 ℃, obtains placenta cDNA.

Recombinant expressed and the purifying of embodiment 2 MAGED1 albumen

In this embodiment, be template with the cDNA of the reverse transcription among the embodiment 1, increase with the PCR Oligonucleolide primers of 5 ' and 3 ' following end of sequence, obtain people MAGED1 DNA as inserting fragment.

5 ' end Oligonucleolide primers sequence is: 5 '-CAGGAGCTCGCTCAGAAAATGGAC-3 ' (SEQ ID NO:4).This primer contains the restriction enzyme site of SacI restriction enzyme, is the part coded sequence that does not contain signal peptide sequence after this restriction enzyme site;

3 ' end primer sequence is: 5 '-TCCCTCGAGTCACTCAACCCAGAA-3 ' (SEQ ID NO:5).This primer contains the part coded sequence of restriction enzyme site, translation termination and the people MAGED1 of XhoI restriction enzyme.

Add 6His tag sequence at pET21a+ carrier (available from Novagen company) N end, formed pT ₇470 carriers (structure is seen Fig. 3).

People MAGED1 albumen cDNA PCR product purification is after the SacI/XhoI double digestion, with the pT of double digestion ₇470 carriers are connected to form carrier pT ₇470-MAGED1 also is converted into competence Escherichia coli HMS174 (DE3) (Novagen company), and the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator kit, PE company).Order-checking confirms to insert complete MAGED1 coded sequence.

Choosing the positive escherichia coli cloning of expressing MAGED1 is inoculated in the 10ml LB nutrient culture media, 37 ℃ of 300rpm shaken cultivation are spent the night, be diluted at 1: 100 and be inoculated in LB nutrient culture media shaken cultivation 2.5hr, add behind the 100mM IPTG to 0.1mM 37 ℃ and induce 2-3hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml sample-loading buffer (0.5M NaCl on ice, 20mM imidazoles 2.7mM KCl, 10.1mM Na ₂HPO ₄, 1.8mM KH ₂PO ₄, pH8.0) resuspended, ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml Ni ²⁺Metal-chelating Sepharose 4B chromatographic column after sample-loading buffer fully washs, adds 500ul imidazoles elution buffer (0.5M NaCl, 500mM imidazoles 2.7 mM KCl, 10.1mM Na ₂HPO ₄, 1.8mM KH ₂PO ₄, pH8.0) room temperature leaves standstill after 30 minutes and collects eluent, repeats wash-out 2-3 time, obtains people MAGED1 albumen.

The result is shown in Figure 1A, and the molecular weight of MAGED1 albumen is about 86.2Kda, conforms to predicted value.

The generation of embodiment 3 anti-MAGED1 protein antibodies

The recombined human MAGED1 albumen that obtains among the embodiment 2 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.With the albumen of 150 μ g/0.2ml emulsifications, rabbit (new zealand rabbit) is carried out intraperitoneal injection.After 14 days,, rabbit (new zealand rabbit) is carried out intraperitoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast idiosyncrasy that obtains is active to be assessed in the ability of external precipitation people MAGED1 protein gene translation product with it.

Found that antibody can combine (Figure 1B) with MAGED1 albumen specifically.

The detection of embodiment 4:MAGED1 in hepatitis, hepatocarcinoma patient serum

Detect MAGED1 albumen among the patients serum with Wester Blot.

Sample preparation: blood serum sample 2ul, 6 * sample preparation liquid 1.67ul, H ₂O 6.33ul.100 ℃, 3 minutes.High speed centrifugation (13,000rpm, 3min).Get sample on the supernatant.

The SDS-PAGE electrophoretic analysis: 12% separation gel, 4% concentrates glue.

Electrotransfer (wet type) is to protran film (Schleicher ﹠amp; Schuell): 400mA 3 hours.

After film is used the PBST rinsing, at room temperature with the PBST sealing that contains 5% skimmed milk power 4 hours.Add the anti-MAGED1 polyclonal antibody of rabbit (1: 500 dilution), 4 ℃ 16 hours.Add the mouse-anti rabbit antibody (Invitrogen, dilution in 1: 2000) that has horseradish peroxidase, room temperature was placed 1 hour.After Super Signal West FemtoMaximum Sensitivity Substrate (PIERCE, horseradish peroxidase substrate) processing, press the X-ray sheet.

The result as shown in Figure 2.Detected hepatocarcinoma patient serum 21 examples altogether, wherein 11 examples detect MAGED1 albumen (positive rate 52%); Hepatitis patient serum's 22 examples of normal human serum 12 examples, the HBV positive, the MAGED1 Protein Detection is all negative.This shows that MAGED1 is a kind of liver cancer marker of special secretion.

Embodiment 5

Detection kit

In the present embodiment, preparation detects the diagnostic kit of MAGED1, this kit contain 200 microlitre embodiment, 3 preparations anti-MAGED1 specific antisera and describe and how to use kit to detect the illustrative material of MAGED1.Kit also can contain one or more in the following group: be used to assist the various labels or the labelled reagent that detect; The reagent that is used to hybridize (comprising damping fluid etc.); Sampling apparatus comprises that fine needle is first-class; And positive and negative hybridization contrast etc.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Xinshijie Gene Techn Development Co., Ltd.

＜120〉as relevant secretory protein of the tumour of liver cancer marker and uses thereof

<130>035647

<160>5

<170>PatentIn?version?3.1

<210>1

<211>2745

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(165)..(2498)

<223>

<400>1

gccgcgctgg?cattttctcc?tggacaagga?gagagtgcgg?ctgctgagag?ccgagcccag 60

caatcccgat?cctctgagtc?gtgaagaagg?gaggcagcga?gggggttggg?gttggggcct 120

gaggcaagcc?cccaggctcc?gctcttgcca?gagggacagg?agcc?atg?gct?cag?aaa 176

Met?Ala?Gln?Lys

1

atg?gac?tgt?ggt?gcg?ggc?ctc?ctc?ggc?ttc?cag?gct?gag?gcc?tcc?gta 224

Met?Asp?Cys?Gly?Ala?Gly?Leu?Leu?Gly?Phe?Gln?Ala?Glu?Ala?Ser?Val

5 10 15 20

gaa?gac?agc?gcc?ttg?ctt?atg?cag?acc?ttg?atg?gag?gcc?atc?cag?atc 272

Glu?Asp?Ser?Ala?Leu?Leu?Met?Gln?Thr?Leu?Met?Glu?Ala?Ile?Gln?Ile

25 30 35

tca?gag?gct?cca?cct?act?aac?cag?gcc?acc?gca?gct?gct?agt?ccc?cag 320

Ser?Glu?Ala?Pro?Pro?Thr?Asn?Gln?Ala?Thr?Ala?Ala?Ala?Ser?Pro?Gln

40 45 50

agt?tca?cag?ccc?cca?act?gcc?aat?gag?atg?gct?gac?att?cag?gtt?tca 368

Ser?Ser?Gln?Pro?Pro?Thr?Ala?Asn?Glu?Met?Ala?Asp?Ile?Gln?Val?Ser

55 60 65

gca?gct?gcc?gct?agg?cct?aag?tca?gcc?ttt?aaa?gtc?cag?aat?gcc?acc 416

Ala?Ala?Ala?Ala?Arg?Pro?Lys?Ser?Ala?Phe?Lys?Val?Gln?Asn?Ala?Thr

70 75 80

aca?aaa?ggc?cca?aat?ggt?gtc?tat?gat?ttc?tct?cag?gct?cat?aat?gcc 464

Thr?Lys?Gly?Pro?Asn?Gly?Val?Tyr?Asp?Phe?Ser?Gln?Ala?His?Asn?Ala

85 90 95 100

aag?gat?gtg?ccc?aac?acg?cag?ccc?aag?gca?gcc?ttt?aag?tcc?caa?aat 512

Lys?Asp?Val?Pro?Asn?Thr?Gln?Pro?Lys?Ala?Ala?Phe?Lys?Ser?Gln?Asn

105 110 115

gct?acc?cca?aag?ggt?cca?aat?gct?gcc?tat?gat?ttt?tcc?cag?gca?gca 560

Ala?Thr?Pro?Lys?Gly?Pro?Asn?Ala?Ala?Tyr?Asp?Phe?Ser?Gln?A1a?Ala

120 125 130

acc?act?ggt?gag?tta?gct?gct?aac?aag?tct?gag?atg?gcc?ttc?aag?gcc 608

Thr?Thr?Gly?Glu?Leu?Ala?Ala?Asn?Lys?Ser?Glu?Met?Ala?Phe?Lys?Ala

135 140 145

cag?aat?gcc?act?act?aaa?gtg?ggc?cca?aat?gcc?acc?tac?aat?ttc?tct 656

Gln?Asn?Ala?Thr?Thr?Lys?Val?Gly?Pro?Asn?Ala?Thr?Tyr?Asn?Phe?Ser

150 155 160

cag?tct?ctc?aat?gcc?aat?gac?ctg?gcc?aac?agc?agg?cct?aag?acc?cct 704

Gln?Ser?Leu?Asn?Ala?Asn?Asp?Leu?Ala?Asn?Ser?Arg?Pro?Lys?Thr?Pro

165 170 175 180

ttc?aag?gct?tgg?aat?gat?acc?act?aag?gcc?cca?aca?gct?gat?acc?cag 752

Phe?Lys?Ala?Trp?Asn?Asp?Thr?Thr?Lys?Ala?Pro?Thr?Ala?Asp?Thr?Gln

185 190 195

acc?cag?aat?gta?aat?cag?gcc?aaa?atg?gcc?act?tcc?cag?gct?gac?ata 800

Thr?Gln?Asn?Val?Asn?Gln?Ala?Lys?Met?Ala?Thr?Ser?Gln?Ala?Asp?Ile

200 205 210

gag?acc?gac?cca?ggt?atc?tct?gaa?cct?gac?ggt?gca?act?gca?cag?aca 848

Glu?Thr?Asp?Pro?Gly?Ile?Ser?Glu?Pro?Asp?Gly?Ala?Thr?Ala?Gln?Thr

215 220 225

tca?gca?gat?ggt?tcc?cag?gct?cag?aat?ctg?gag?tcc?cgg?aca?ata?att 896

Ser?Ala?Asp?Gly?Ser?Gln?Ala?Gln?Asn?Leu?Glu?Ser?Arg?Thr?Ile?Ile

230 235 240

cgg?ggc?aag?agg?acc?cgc?aag?att?aat?aac?ttg?aat?gtt?gaa?gag?aac 944

Arg?Gly?Lys?Arg?Thr?Arg?Lys?Ile?Asn?Asn?Leu?Asn?Val?Glu?Glu?Asn

245 250 255 260

agc?agt?ggg?gat?cag?agg?cgg?gcc?cca?ctg?gct?gca?ggg?acc?tgg?agg 992

Ser?Ser?Gly?Asp?Gln?Arg?Arg?Ala?Pro?Leu?Ala?Ala?Gly?Thr?Trp?Arg

265 270 275

tct?gca?cca?gtt?cca?gtg?acc?act?cag?aac?cca?cct?ggc?gca?ccc?ccc 1040

Ser?Ala?Pro?Val?Pro?Val?Thr?Thr?Gln?Asn?Pro?Pro?Gly?Ala?Pro?Pro

280 285 290

aat?gtg?ctc?tgg?cag?acg?cca?ttg?gct?tgg?cag?aac?ccc?tca?ggc?tgg 1088

Asn?Val?Leu?Trp?Gln?Thr?Pro?Leu?Ala?Trp?Gln?Asn?Pro?Ser?Gly?Trp

295 300 305

caa?aac?cag?aca?gcc?agg?cag?acc?cca?cca?gca?cgt?cag?agc?cct?cca 1136

Gln?Asn?Gln?Thr?Ala?Arg?Gln?Thr?Pro?Pro?Ala?Arg?Gln?Ser?Pro?Pro

310 315 320

gct?agg?cag?acc?cca?cca?gcc?tgg?cag?aac?cca?gtc?gct?tgg?cag?aac 1184

Ala?Arg?Gln?Thr?Pro?Pro?Ala?Trp?Gln?Asn?Pro?Val?Ala?Trp?Gln?Asn

325 330 335 340

cca?gtg?att?tgg?cca?aac?cca?gta?atc?tgg?cag?aac?cca?gtg?atc?tgg 1232

Pro?Val?Ile?Trp?Pro?Asn?Pro?Val?Ile?Trp?Gln?Asn?Pro?Val?Ile?Trp

345 350 355

cca?aac?ccc?att?gtc?tgg?ccc?ggc?cct?gtt?gtc?tgg?ccg?aat?cca?ctg 1280

Pro?Asn?Pro?Ile?Val?Trp?Pro?Gly?Pro?Val?Val?Trp?Pro?Asn?Pro?Leu

360 365 370

gcc?tgg?cag?aat?cca?cct?gga?tgg?cag?act?cca?cct?gga?tgg?cag?acc 1328

Ala?Trp?Gln?Asn?Pro?Pro?Gly?Trp?Gln?Thr?Pro?Pro?Gly?Trp?Gln?Thr

375 380 385

cca?ccg?ggc?tgg?cag?ggt?cct?cca?gac?tgg?caa?ggt?cct?cct?gac?tgg 1376

Pro?Pro?Gly?Trp?Gln?Gly?Pro?Pro?Asp?Trp?Gln?Gly?Pro?Pro?Asp?Trp

390 395 400

ccg?cta?cca?ccc?gac?tgg?cca?ctg?cca?cct?gat?tgg?cca?ctt?ccc?act 1424

Pro?Leu?Pro?Pro?Asp?Trp?Pro?Leu?Pro?Pro?Asp?Trp?Pro?Leu?Pro?Thr

405 410 415 420

gac?tgg?cca?cta?cca?cct?gac?tgg?atc?ccc?gct?gat?tgg?cca?att?cca 1472

Asp?Trp?Pro?Leu?Pro?Pro?Asp?Trp?Ile?Pro?Ala?Asp?Trp?Pro?Ile?Pro

425 430 435

cct?gac?tgg?cag?aac?ctg?cgc?ccc?tcg?cct?aac?ctg?cgc?cct?tct?ccc 1520

Pro?Asp?Trp?Gln?Asn?Leu?Arg?Pro?Ser?Pro?Asn?Leu?Arg?Pro?Ser?Pro

440 445 450

aac?tcg?cgt?gcc?tca?cag?aac?cca?ggt?gct?gca?cag?ccc?cga?gat?gtg 1568

Asn?Ser?Arg?Ala?Ser?Gln?Asn?Pro?Gly?Ala?Ala?Gln?Pro?Arg?Asp?Val

455 460 465

gcc?ctt?ctt?cag?gaa?aga?gca?aat?aag?ttg?gtc?aag?tac?ttg?atg?ctt 1616

Ala?Leu?Leu?Gln?Glu?Arg?Ala?Asn?Lys?Leu?Val?Lys?Tyr?Leu?Met?Leu

470 475 480

aag?gac?tac?aca?aag?gtg?ccc?atc?aag?cgc?tca?gaa?atg?ctg?aga?gat 1664

Lys?Asp?Tyr?Thr?Lys?Val?Pro?Ile?Lys?Arg?Ser?Glu?Met?Leu?Arg?Asp

485 490 495 500

atc?atc?cgt?gaa?tac?act?gat?gtt?tat?cca?gaa?atc?att?gaa?cgt?gca 1712

Ile?Ile?Arg?Glu?Tyr?Thr?Asp?Val?Tyr?Pro?Glu?Ile?Ile?Glu?Arg?Ala

505 510 515

tgc?ttt?gtc?cta?gag?aag?aaa?ttt?ggg?att?caa?ctg?aaa?gaa?att?gac 1760

Cys?Phe?Val?Leu?Glu?Lys?Lys?Phe?Gly?Ile?Gln?Leu?Lys?Glu?Ile?Asp

520 525 530

aaa?gaa?gaa?cac?ctg?tat?att?ctc?atc?agt?acc?ccc?gag?tcc?ctg?gct 1808

Lys?Glu?Glu?His?Leu?Tyr?Ile?Leu?Ile?Ser?Thr?Pro?Glu?Ser?Leu?Ala

535 540 545

ggc?ata?ctg?gga?acg?acc?aaa?gac?aca?ccc?aag?ctc?ggt?ctc?ctc?ttg 1856

Gly?Ile?Leu?Gly?Thr?Thr?Lys?Asp?Thr?Pro?Lys?Leu?Gly?Leu?Leu?Leu

550 555 560

gtg?att?ctg?ggt?gtc?atc?ttc?atg?aat?ggc?aac?cgt?gcc?agt?gag?gct 1904

Val?Ile?Leu?Gly?Val?Ile?Phe?Met?Asn?Gly?Asn?Arg?Ala?Ser?Glu?Ala

565 570 575 580

gtc?ctc?tgg?gag?gca?cta?cgc?aag?atg?gga?ctg?cgt?cct?ggg?gtg?aga 1952

Val?Leu?Trp?Glu?Ala?Leu?Arg?Lys?Met?Gly?Leu?Arg?Pro?Gly?Val?Arg

585 590 595

cat?ccc?ctc?ctt?gga?gat?cta?agg?aaa?ctt?ctc?acc?tat?gag?ttt?gta 2000

His?Pro?Leu?Leu?Gly?Asp?Leu?Arg?Lys?Leu?Leu?Thr?Tyr?Glu?Phe?Val

600 605 610

aag?cag?aaa?tac?ctg?gac?tac?aga?cga?gtg?ccc?aac?agc?aac?ccc?ccg 2048

Lys?Gln?Lys?Tyr?Leu?Asp?Tyr?Arg?Arg?Val?Pro?Asn?Ser?Asn?Pro?Pro

615 620 625

gag?tat?gag?ttc?ctc?tgg?ggc?ctc?cgt?tcc?tac?cat?gag?act?agc?aag 2096

Glu?Tyr?Glu?Phe?Leu?Trp?Gly?Leu?Arg?Ser?Tyr?His?Glu?Thr?Ser?Lys

630 635 640

atg?aaa?gtg?ctg?aga?ttc?att?gca?gag?gtt?cag?aaa?aga?gac?cct?cgt 2144

Met?Lys?Val?Leu?Arg?Phe?Ile?Ala?Glu?Val?Gln?Lys?Arg?Asp?Pro?Arg

645 650 655 660

gac?tgg?act?gca?cag?ttc?atg?gag?gct?gca?gat?gag?gcc?ttg?gat?gct 2192

Asp?Trp?Thr?Ala?Gln?Phe?Met?Glu?Ala?Ala?Asp?Glu?Ala?Leu?Asp?Ala

665 670 675

ctg?gat?gct?gct?gca?gct?gag?gcc?gaa?gcc?agg?gct?gaa?gca?aga?acc 2240

Leu?Asp?Ala?Ala?Ala?Ala?Glu?Ala?Glu?Ala?Arg?Ala?Glu?Ala?Arg?Thr

680 685 690

cgc?atg?gga?att?gga?gat?gag?gct?gtg?tct?ggg?ccc?tgg?agc?tgg?gat 2288

Arg?Met?Gly?Ile?Gly?Asp?Glu?Ala?Val?Ser?Gly?Pro?Trp?Ser?Trp?Asp

695 700 705

gac?att?gag?ttt?gag?ctg?ctg?acc?tgg?gat?gag?gaa?gga?gat?ttt?gga 2336

Asp?Ile?Glu?Phe?Glu?Leu?Leu?Thr?Trp?Asp?Glu?Glu?Gly?Asp?Phe?Gly

710 715 720

gat?ccc?tgg?tcc?aga?att?cca?ttt?acc?ttc?tgg?gcc?aga?tac?cac?cag 2384

Asp?Pro?Trp?Ser?Arg?Ile?Pro?Phe?Thr?Phe?Trp?Ala?Arg?Tyr?His?Gln

725 730 735 740

aat?gcc?cgc?tcc?aga?ttc?cct?cag?acc?ttt?gcc?ggt?ccc?att?att?ggt 2432

Asn?Ala?Arg?Ser?Arg?Phe?Pro?Gln?Thr?Phe?Ala?Gly?Pro?Ile?Ile?Gly

745 750 755

cct?ggt?ggt?aca?gcc?agt?gcc?aac?ttc?gct?gcc?aac?ttt?ggt?gcc?att 2480

Pro?Gly?Gly?Thr?Ala?Ser?Ala?Asn?Phe?Ala?Ala?Asn?Phe?Gly?Ala?Ile

760 765 770

ggt?ttc?ttc?tgg?gtt?gag?tgagatgttg?gatattgcta?tcaatcgcag 2528

Gly?Phe?Phe?Trp?Val?Glu

775

tagtctttcc?cctgtgtgag?gctgaagcct?cagattcctt?ctaaacacag?ctatctagag 2588

agccacatcc?tgttgactga?aagtggcatg?caagataaat?ttatttgctg?ttccttgtct 2648

actgcttttt?ttccccttgt?gtgctgtcaa?gttttggtat?cagaaataaa?cattgaaatt 2708

gcaaagtgaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaa 2745

<210>2

<211>778

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met?Ala?Gln?Lys?Met?Asp?Cys?Gly?Ala?Gly?Leu?Leu?Gly?Phe?Gln?Ala

1 5 10 15

Glu?Ala?Ser?Val?Glu?Asp?Ser?Ala?Leu?Leu?Met?Gln?Thr?Leu?Met?Glu

20 25 30

Ala?Ile?Gln?Ile?Ser?Glu?Ala?Pro?Pro?Thr?Asn?Gln?Ala?Thr?Ala?Ala

35 40 45

Ala?Ser?Pro?Gln?Ser?Ser?Gln?Pro?Pro?Thr?Ala?Asn?Glu?Met?Ala?Asp

50 55 60

Ile?Gln?Val?Ser?Ala?Ala?Ala?Ala?Arg?Pro?Lys?Ser?Ala?Phe?Lys?Val

65 70 75 80

Gln?Asn?Ala?Thr?Thr?Lys?Gly?Pro?Asn?Gly?Val?Tyr?Asp?Phe?Ser?Gln

85 90 95

Ala?His?Asn?Ala?Lys?Asp?Val?Pro?Asn?Thr?Gln?Pro?Lys?Ala?Ala?Phe

100 105 110

Lys?Ser?Gln?Asn?Ala?Thr?Pro?Lys?Gly?Pro?Asn?Ala?Ala?Tyr?Asp?Phe

115 120 125

Ser?Gln?Ala?Ala?Thr?Thr?Gly?Glu?Leu?Ala?Ala?Asn?Lys?Ser?Glu?Met

130 135 140

Ala?Phe?Lys?Ala?Gln?Asn?Ala?Thr?Thr?Lys?Val?Gly?Pro?Asn?Ala?Thr

145 150 155 160

Tyr?Asn?Phe?Ser?Gln?Ser?Leu?Asn?Ala?Asn?Asp?Leu?Ala?Asn?Ser?Arg

165 170 175

Pro?Lys?Thr?Pro?Phe?Lys?Ala?Trp?Asn?Asp?Thr?Thr?Lys?Ala?Pro?Thr

180 185 190

Ala?Asp?Thr?Gln?Thr?Gln?Asn?Val?Asn?Gln?Ala?Lys?Met?Ala?Thr?Ser

195 200 205

Gln?Ala?Asp?Ile?Glu?Thr?Asp?Pro?Gly?Ile?Ser?Glu?Pro?Asp?Gly?Ala

210 215 220

Thr?Ala?Gln?Thr?Ser?Ala?Asp?Gly?Ser?Gln?Ala?Gln?Asn?Leu?Glu?Ser

225 230 235 240

Arg?Thr?Ile?Ile?Arg?Gly?Lys?Arg?Thr?Arg?Lys?Ile?Asn?Asn?Leu?Asn

245 250 255

Val?Glu?Glu?Asn?Ser?Ser?Gly?Asp?Gln?Arg?Arg?Ala?Pro?Leu?Ala?Ala

260 265 270

Gly?Thr?Trp?Arg?Ser?Ala?Pro?Val?Pro?Val?Thr?Thr?Gln?Asn?Pro?Pro

275 280 285

Gly?Ala?Pro?Pro?Asn?Val?Leu?Trp?Gln?Thr?Pro?Leu?Ala?Trp?Gln?Asn

290 295 300

Pro?Ser?Gly?Trp?Gln?Asn?Gln?Thr?Ala?Arg?Gln?Thr?Pro?Pro?Ala?Arg

305 310 315 320

Gln?Ser?Pro?Pro?Ala?Arg?Gln?Thr?Pro?Pro?Ala?Trp?Gln?Asn?Pro?Val

325 330 335

Ala?Trp?Gln?Asn?Pro?Val?Ile?Trp?Pro?Asn?Pro?Val?Ile?Trp?Gln?Asn

340 345 350

Pro?Val?Ile?Trp?Pro?Asn?Pro?Ile?Val?Trp?Pro?Gly?Pro?Val?Val?Trp

355 360 365

Pro?Asn?Pro?Leu?Ala?Trp?Gln?Asn?Pro?Pro?Gly?Trp?Gln?Thr?Pro?Pro

370 375 380

Gly?Trp?Gln?Thr?Pro?Pro?Gly?Trp?Gln?Gly?Pro?Pro?Asp?Trp?Gln?Gly

385 390 395 400

Pro?Pro?Asp?Trp?Pro?Leu?Pro?Pro?Asp?Trp?Pro?Leu?Pro?Pro?Asp?Trp

405 410 415

Pro?Leu?Pro?Thr?Asp?Trp?Pro?Leu?Pro?Pro?Asp?Trp?Ile?Pro?Ala?Asp

420 425 430

Trp?Pro?Ile?Pro?Pro?Asp?Trp?Gln?Asn?Leu?Arg?Pro?Ser?Pro?Asn?Leu

435 440 445

Arg?Pro?Ser?Pro?Asn?Ser?Arg?Ala?Ser?Gln?Asn?Pro?Gly?Ala?Ala?Gln

450 455 460

Pro?Arg?Asp?Val?Ala?Leu?Leu?Gln?Glu?Arg?Ala?Asn?Lys?Leu?Val?Lys

465 470 475 480

Tyr?Leu?Met?Leu?Lys?Asp?Tyr?Thr?Lys?Val?Pro?Ile?Lys?Arg?Ser?Glu

485 490 495

Met?Leu?Arg?Asp?Ile?Ile?Arg?Glu?Tyr?Thr?Asp?Val?Tyr?Pro?Glu?Ile

500 505 510

Ile?Glu?Arg?Ala?Cys?Phe?Val?Leu?Glu?Lys?Lys?Phe?Gly?Ile?Gln?Leu

515 520 525

Lys?Glu?Ile?Asp?Lys?Glu?Glu?His?Leu?Tyr?Ile?Leu?Ile?Ser?Thr?Pro

530 535 540

Glu?Ser?Leu?Ala?Gly?Ile?Leu?Gly?Thr?Thr?Lys?Asp?Thr?Pro?Lys?Leu

545 550 555 560

Gly?Leu?Leu?Leu?Val?Ile?Leu?Gly?Val?Ile?Phe?Met?Asn?Gly?Asn?Arg

565 570 575

Ala?Ser?Glu?Ala?Val?Leu?Trp?Glu?Ala?Leu?Arg?Lys?Met?Gly?Leu?Arg

580 585 590

Pro?Gly?Val?Arg?His?Pro?Leu?Leu?Gly?Asp?Leu?Arg?Lys?Leu?Leu?Thr

595 600 605

Tyr?Glu?Phe?Val?Lys?Gln?Lys?Tyr?Leu?Asp?Tyr?Arg?Arg?Val?Pro?Asn

610 615 620

Ser?Asn?Pro?Pro?Glu?Tyr?Glu?Phe?Leu?Trp?Gly?Leu?Arg?Ser?Tyr?His

625 630 635 640

Glu?Thr?Ser?Lys?Met?Lys?Val?Leu?Arg?Phe?Ile?Ala?Glu?Val?Gln?Lys

645 650 655

Arg?Asp?Pro?Arg?Asp?Trp?Thr?Ala?Gln?Phe?Met?Glu?Ala?Ala?Asp?Glu

660 665 670

Ala?Leu?Asp?Ala?Leu?Asp?Ala?Ala?Ala?Ala?Glu?Ala?Glu?Ala?Arg?Ala

675 680 685

Glu?Ala?Arg?Thr?Arg?Met?Gly?Ile?Gly?Asp?Glu?Ala?Val?Ser?Gly?Pro

690 695 700

Trp?Ser?Trp?Asp?Asp?Ile?Glu?Phe?Glu?Leu?Leu?Thr?Trp?Asp?Glu?Glu

705 710 715 720

Gly?Asp?Phe?Gly?Asp?Pro?Trp?Ser?Arg?Ile?Pro?Phe?Thr?Phe?Trp?Ala

725 730 735

Arg?Tyr?His?Gln?Asn?Ala?Arg?Ser?Arg?Phe?Pro?Gln?Thr?Phe?Ala?Gly

740 745 750

Pro?Ile?Ile?Gly?Pro?Gly?Gly?Thr?Ala?Ser?Ala?Asn?Phe?Ala?Ala?Asn

755 760 765

Phe?Gly?Ala?Ile?Gly?Phe?Phe?Trp?Val?Glu

770 775

<210>3

<211>50

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

gagagagaga?gagagagaga?actagtctcg?agtttttttt?tttttttttt 50

<210>4

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

caggagctcg?ctcagaaaat?ggac 24

<210>5

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

tccctcgagt?cactcaaccc?agaa 24

Claims (10)

1. a kit that detects liver cancer is characterized in that, it contains antibody and the instructions that combines with the MAGED1 protein-specific.

2. kit as claimed in claim 1 is characterized in that, the monoclonal antibody of described antibody.

3. kit as claimed in claim 1 is characterized in that, the polyclonal antibody of described antibody.

4. kit as claimed in claim 1 is characterized in that described kit also contains positive control and negative control.

5. kit as claimed in claim 1 is characterized in that described kit also contains sampling apparatus.

6. protein-chip that detects cancer, it comprises that substrate and point sample at the specific antibody of on-chip cancer associated protein, is characterized in that, the specific antibody of described cancer associated protein comprises the specific antibody of anti-MAGED1.

7. protein-chip as claimed in claim 6, it is characterized in that the specific antibody of described cancer associated protein also comprises the antibody of anti-following antigen: pTEN, p21, p27, p73, p15, p53, RB1, APC, nm23, P16, MXR7, IGF-II, TGF α, HGF-R, c-erbB-1, Ras, Raf, c-myc, c-ets-2.

8. whether there is the method for MAGED1 albumen in the test sample, it is characterized in that, comprise step:

The antibody of sample with the combination of MAGED1 protein-specific is contacted, observes whether form antibody complex, formed antibody complex and just represented to exist in the sample MAGED1 albumen,

Wherein, described sample is a blood sample.

9. method as claimed in claim 8 is characterized in that, described blood sample is people's a blood sample.

10. a pharmaceutical composition is characterized in that, it contains the MAGED1 antagonist and the pharmaceutically acceptable carrier of safe and effective amount.

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