CN110194801A

CN110194801A - A kind of fusion protein and polyclonal antibody and its application

Info

Publication number: CN110194801A
Application number: CN201810187751.7A
Authority: CN
Inventors: 张灏
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-02-26
Filing date: 2018-03-07
Publication date: 2019-09-03
Anticipated expiration: 2038-03-07
Also published as: CN110194801B

Abstract

The invention discloses a kind of fusion protein of tumor specific expression, amino acid sequence is as shown in SEQ ID NO.1 or its coded sequence is as shown in SEQ ID NO.2.The invention also discloses a kind of polyclonal antibody, antigen is at least segment polypeptide in fusion protein or fusion protein；The invention also discloses the preparation method and application of the polyclonal antibody.The fusion protein ASTN2-PAPPA found for the first time in the present invention is the fusion protein occurred in malignant tumour, research shows that growth, the transfer of itself and tumour have substantial connection, the ASTN2-PAPPA polyclonal antibody that the present invention specially designs can identify the fusion protein, provide reliable tool to study protein level and the molecular mechanism of the fusion protein；ASTN2-PAPPA is as the fusion protein in close relations with tumor development, it can be used as molecular marker and therapy target in terms of the diagnosing and treating of tumour, the polyclonal antibody provided by the present invention for ASTN2-PAPPA fusion protein provides possibility for the diagnosis in future and Antybody therapy.

Description

A kind of fusion protein and polyclonal antibody and its application

Technical field

The present invention relates to antibody production techniques field, especially a kind of fusion protein and polyclonal antibody and its application.

Background technique

Cancer is mankind's number one killer, and previous cancer gives people and feels to be incurable disease, but with the development of medicine, passes through hand Art, radiotherapy, chemotherapy and target drug therapy etc. can effectively extend patient's service life.There are many reason of cancer occurs, dangerous Factor is also varied, such as chemical factor, physical factor, biological factor.

It not only plays an important role in tumour generation in cancer research discovery fusion protein at present, but also there is huge turn Change meaning, can be used as therapy target and molecular marker.The mechanism that fusion protein generates includes: 1) various chromosome variations Fusion protein caused by fusion caused by (including DNA insertion, amplification, missing, rearrangement, indexing)；2) various RNA are abnormal The fusion protein that fusion rna caused by montage is translated into.

Foremost fusion protein is the BCR-ABL albumen generated by fusion Philadelphia chromosome.This discovery starts from The last century 60's, the Philadelphia chromosome found in chronic myelogenous leukemia patient have pulled open fusion protein and cancer Study prelude, BCR-ABL fusion extremely albumen is present in 95% chronic myelocytic leukemia, BCR-ABL not only at For the important diagnostic marker of CML, and its inhibitor Gleevec (Gleevec) is the healing of the once high CML of lethality Rate improves 61%.

Fusion protein is had found in kinds cancer at present, and develops the clinical medicine for fusion protein. Fusion protein can promote the occurrence and development of tumour, and can be used as the molecular diagnosis and therapeutic targets of tumour.As RNA is sequenced deeply The development of technology, more and more fusion rnas and its fusion protein product are gradually found.The detection method of fusion protein at present Mainly have:

1) in DNA level detection fusion gene.Fluorescence in situ hybridization (FISH) is chromosome in current detection tumor tissues Reset one of most effective diagnostic techniques.FDA approval has been obtained, has been marked as the gold for detecting ALK fusion gene in clinical trial Standard, the technology are also the common method of ROS1 fusion clinical detection.For example, FISH fracture probe is normal ALK or ROS1 The end 5'- with the 3'- difference label of gene has the probe with red fluorescence, when there are gene rearrangement, green and red letter Number because recombinate fusion partners displacement due to " fracture " is revealed as separating farther away monochrome signal.As a kind of conventional screening side Method, FISH testing cost is relatively high, needs special equipment, expensive reagents, and the processing time is longer, testing result be difficult to interpretation, The deficiencies of subjectivity is strong, there is also some limitations in practical application.

2) in rna level detection fusion RNA.RT-PCR technology is simple and quick accurate, can specify Gene Fusion type, is applicable in In microcomponent's sample, thus RT-PCR technology is being screened and is being confirmed in lung cancer genetic recombination with unique advantage.But RT- PCR is higher to DNA quality requirement in tissue, false positive as a result usually occurs.

3) in protein level detection fusion albumen.Partial fusion gene codified protein plays the occurrence and development of tumour To important role.The protein of some fusions and its coding can be used as the marker of instruction cancer occurrence and development. So needing to study the expression of its gene level and protein level simultaneously in the research process of the fusion of cancer.Fusion Gene as product completely new in an organism, the protein of coding be also it is unprecedented, so a specific needle It is extremely important to the antibody of fusion coding protein for the research of fusion.Immunohistochemistry skill Art (IHC) is specific antigen in the antibody position tissue sample marked with probe.The technology is widely used to clinic, and It is at low cost, easy to be quick, accurate.Recently the detection of IHC colouring method ALK, the ROS1 carried out using high sensitive high specific antibody Isogenic rearrangement, as a result, it has been found that accuracy can reach 100%, specificity up to 99% compared with being broken FISH technology.

It can be seen that fusion protein specific antibody is for pushing the development of fusion protein research to have irreplaceable work With.

Summary of the invention

Present inventor has found a kind of fusion protein during studying tumour mechanism and treatment method for the first time ASTN2-PAPPA is the fusion protein after the fusion rna translation that RNA aberrant splicing is formed.Inventor sends out in the course of the research Existing ASTN2-PAPPA is specific expressed in the cancer of the esophagus, therefore can be used as tumor markers and therapy target.

Currently, having the antibody of the albumen for ASTN2 and PAPPA gene coding, but without being directed to two Gene Fusions The antibody of encoded albumin A STN2-PAPPA (abbreviation A-P) later.

The present invention designs for a kind of fusion ASTN2-PAPPA of specific amplification in tumour and prepares specific needle To the polyclonal antibody of the fusion protein, for the tool provided convenience of research of the fusion and effective means.Facing Bed conversion aspect, ASTN2-PAPPA can not only be converted into molecular diagnostic markers, can also develop for ASTN2-PAPPA Specific antibodies medicine be used for clinical tumor immunization therapy.

ASTN2-PAPPA is a completely new fusion and coding protein, is risen in the occurrence and development of cancer To important function, there is presently no the specific antibodies for being directed to A-P, limit relevant tumor research, also limit clinical phase The development and application of the tumor markers and antibody drug of pass.

To solve the above-mentioned problems, the technical solution that the present invention takes includes the following aspects:

As the first aspect of the invention, the present invention provides a kind of fusion protein (i.e. ASTN2-PAPPA), the fusion The amino acid sequence of albumen is as shown in SEQ ID NO.1 or the coded sequence of the fusion protein is as shown in SEQ ID NO.2.

As another aspect of the present invention, the present invention provides a kind of polyclonal antibody, and the antigen of the antibody is above-mentioned Fusion protein or above-mentioned fusion protein in an at least segment polypeptide.

Preferably, the polypeptide covers the position of fusion of the fusion protein.

As another aspect of the present invention, the present invention provides the preparation method of above-mentioned antibody, includes the following steps:

(1) Antigen Expression Vectors are constructed, wherein the nucleotide sequence for expressing antigen covers the fusion of above-mentioned fusion protein Site or so at least nucleotide sequence of 12bp；

(2) step (1) acquisition carrier is converted to competent cell, with purifying antigen；

(3) the antigen immune rabbit after purification that is obtained with step (2) simultaneously collects serum, obtains the Anti-TNF-α Body.At least four amino acid of position of fusion two sides as a result, the i.e. peptide fragment of at least eight amino acid length guarantee anti-as antigen Original antibody binding specificity.Wherein, immune animal includes but is not limited to rabbit, can also be mouse, rat etc..

Preferably, above-mentioned preparation method further includes step (4): using serum obtained by salting out method purification step (3) to obtain Polyclonal antibody.Wherein the method for purified blood serum includes but is not limited to salting out method.

Preferably, the carrier uses plasmid ppyCAGIP.Wherein, carrier is other than it can be plasmid ppyCAGIP, also It can be other plasmids, as long as antigen described in energy normal expression.

Preferably, the cell is BL21.Wherein, competent cell includes but is not limited to BL21, can also be other thin Born of the same parents, such as DH10Bac etc..

As another aspect of the present invention, the present invention provides above-mentioned polyclonal antibody and detects above-mentioned fusion egg in preparation Application in the kit of bletilla its table level.

As another aspect of the present invention, the present invention provides above-mentioned polyclonal antibody in the drug of preparation treatment tumour Or the application in the above-mentioned fusion protein of research.

As another aspect of the present invention, the present invention provides a kind of drug for treating tumour, and the drug contains above-mentioned Polyclonal antibody.Wherein, the preferred cancer of the esophagus of the tumour.

In conclusion the invention has the benefit that

The fusion protein ASTN2-PAPPA found for the first time in the present invention is the fusion protein occurred in malignant tumour, research Show that growth, the transfer of itself and tumour have substantial connection, the ASTN2-PAPPA polyclonal antibody that the present invention specially designs can reflect The fixed fusion protein, provides reliable tool to study protein level and the molecular mechanism of the fusion protein；

ASTN2-PAPPA is as the fusion protein in close relations with tumor development, in the diagnosing and treating side of tumour Face can be used as molecular marker and therapy target, the Anti-TNF-α provided by the present invention for ASTN2-PAPPA fusion protein Body provides possibility for the diagnosis in future and Antybody therapy.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of ppyCAGIP plasmid；

Fig. 2 is the electrophoretogram of purpose segment；

Fig. 3 is ELISA testing result, wherein (-): negative serum 1:1000OD < 0.2；ELISA titre > 1:64000；

Fig. 4 is the result figure of Western blot experiment detection ASTN2-PAPPA fusion protein；

Fig. 5 is the result figure of ASTN2-PAPPA antibody test tumor cells expression difference；

Fig. 6 is variation diagram of the ASTN2-PAPPA antibody test normal tissue into cancerous tissue conversion process.

Specific embodiment

The present invention relates to oncomolecularbiology field, in particular aspect, the present invention relates to the preparation of antibody and antigen, The specificity analysis of antibody, application of the polyclonal antibody in tumor research and clinical treatment.

The present invention provides the specific antigen of anti-fusion protein ASTN2-PAPPA a kind of and its specificity of polyclonal antibody Analysis method.The antibody specificity is integrated to the integration region of source of people ASTN2-PAPPA fusion protein, rather than combines PAPPA Or the non-fused region of ASTN2.When detecting A-P fusion protein using ASTN2-PAPPA polyclonal antibody of the invention, then say Bright ASTN2 and PAPPA are merged；Fusion protein ASTN2-PAPPA polyclonal antibody of the invention can be used for melting in the future The research and clinical diagnosis and treatment of effect of the conjunction gene in tumour.

To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.The specific embodiment of the application, such as host animal, antibody purification process, cloning vector and sense It is including but not limited to as described below by state cell.Those skilled in the art are without departing from spirit and scope the case where Under, appropriate change can be made to the present invention.In the case where not expressing, used reagent and its dosage unit are all abilities Domain routine.

The discovery of embodiment 1ASTN2-PAPPA fusion protein

Experimental method: collecting the pathological tissue sample of tumour patient, by high throughput sequencing technologies, finds in malignant tumour In, ASTN2 gene (ID:23245) and PAPPA gene (ID:5069) are merged, and new fusion protein ASTN2- is formd PAPPA, and the position merged and its sequence has been determined.

(1) nucleotide sequence of fusion protein ASTN2-PAPPA is as follows:

GATTGATCTACCATGGACTACAAAGACGATGACGACAAGGGGATCATGGCCGCCGCCGGCGCCCGGCTCAGCCCCGG CCCCGGCTCGGGGCTCCGGGGGCGGCCGAGGCTCTGCTTCCACCCGGGGCCGCCGCCACTGCTGCCGCTGCTGCTGC TGTTCCTGCTCCTGCTGCCGCCGCCGCCGCTGCTGGCCGGCGCCACCGCCGCTGCCTCGCGGGAGCCCGACAGCCCG TGCCGGCTGAAGACCGTCACGGTGTCCACACTGCCCGCCCTGCGGGAGAGCGACATCGGCTGGAGCGGCGCCCGCGC CGGGGCCGGGGCTGGGACCGGGGCCGGAGCCGCCGCCGCCGCCGCGTCCCCGGGCTCTCCTGGCTCTGCCGGCACCG CCGCCGAGTCGCGCCTCCTGCTCTTTGTGCGTAACGAGCTGCCGGGGCGCATCGCGGTGCAGGACGACCTGGACAAC ACCGAGCTGCCCTTCTTCACCCTGGAGATGTCTGGCACAGCGGCGGACATCTCGCTGGTGCACTGGAGACAGCAGTG GCTGGAGAATGGCACCTTGTACTTCCACGTCTCCATGAGCAGCTCCGGGCAGCTGGCCCAAGCCACCGCCCCCACTC TCCAGGAGCCCTCGGAGATTGTTGAGGAGCAGATGCACATCCTCCACATTTCTGTGATGGGTGGCCTCATCGCGCTG CTGCTGCTGCTGCTGGTGTTCACCGTGGCGCTGTACGCCCAGCGACGTTGGCAGAAGCGTCGCCGCATCCCCCAGAA GAGCGCAAGCACAGAAGCCACTCATGAGATCCACTACATCCCATCTGTGCTGCTGGGTCCCCAGGCGCGGGAGAGCT TCCGTTCATCCCGGCTGCAAACCCACAATTCCGTCATTGGCGTGCCCATCCGGGAGACTCCCATCCTGGATGACTAT GACTGTGAGGAGGATGAGGAGCCACCTAGGCGGGCCAACCATGTCTCCCGCGAGGACGAGTTTGGCAGCCAGGTGAC CCACACTCTGGACAGTCTGGGACATCCAGGGGAAGAGAAGGTGGACTTTGAGAAGAAAGCAGCAGCTGAGGCGACTC AGGAGACAGTGGAGTCCCTGATGCAGAAGTTCAAGGAGAGTTTCCGCGCTAACACGCCCATCGAGATCGGTCAGCTG CAACCACCCCTGCGCAGCACATCGGCAGGGAAGAGGAAGCGGAGGAGCAAGTCTCGAGGAGGAATCAGCTTTGGGAG AGCCAAGGGGACGTCGGGCTCAGAGGCAGACGATGAAACTCAGCTGACATTCTACACGGAGCAGTACCGCAGTCGCC GCCGCAGCAAAGGTTTGCTGAAAAGCCCAGTGAACAAGACAGCCCTGACACTGATTGCTGTGAGTTCCTGCATCCTG GCCATGGTGTGTGGCAGCCAGATGTCTTGTCCACTCACTGTGAAGGTGACTCTGCATGTGCCCGAGCACTTCATAGC AGATGGAAGCAGCTTCGTGGTGAGTGAAGGGAGCTACCTGGACATCTCCGACTGGTTAAACCCAGCCAAGCTTTCCC TGTATTACCAGATCAATGCCACCTCCCCATGGGTGAGGGACCTCTGTGGACAAAGGACGACAGATGCCTGTGAGCAG CTCTGCGACCCAGAAACCGGAGAGTGCAGCTGTCATGAAGGCTATGCCCCTGACCCTGTTCACAGACACCTGTGTGT GCGCAGTGACTGGGGACAGAGTGAAGGACCTTGGCCCTACACGACACTTGAGAGGGGCTATGATCTGGTGACAGGGG AGCAAGCCCCTGAAAAGATTCTCAGGTCTACTTTCAGCTTGGGCCAAGGCCTCTGGCTTCCTGTCAGCAAAAGCTTT GTGGTTCCGCCTGTGGAGCTGTCCATCAACCCCCTGGCCAGCTGCAAGACCGATGTGCTCGTCACGGAAGACCCTGC AGATGTCAGGGAAGAAGCGATGCTGTCCACATACTTTGAAACCATCAATGACCTGCTGTCTTCCTTCGGGCCAGTTC GTGACTGCTCTCGGAACAATGGGGGCTGCACTCGCAACTTCAAGTGTGTGTCTGACCGGCAGGTGGATTCCTCGGGA TGTGTGTGCCCTGAGGAGCTGAAACCCATGAAGGATGGCTCTGGCTGCTACGACCACTCCAAAGGCATTGACTGCTC TGATGGCTTTAATGGCGGCTGTGAGCAGCTGTGCCTGCAGCAGACGCTGCCCCTGCCCTACGATGCCACTTCGAGCA CCATCTTCATGTTCTGCGGTTGCGTGGAGGAGTACAAACTGGCTCCTGATGGAAAATCCTGCTTAATGCTCTCAGAT GTCTGCGAGGGCCCCAAGTGCCTCAAACCTGACTCCAAATTCAATGATACCCTCTTTGGAGAGATGCTACATGGTTA CAACAACCGGACCCAGCATGTGAACCAAGGCCAAGTCTTCCAGATGACCTTTAGGGAGAACAACTTCATCAAGGACT TTCCCCAGCTGGCCGATGGGCTGTTGGTGATCCCGCTGCCGGTGGAGGAGCAGTGCCGGGGGGTCCTCTCCGAGCCC CTTCCGGACCTCCAACTGCTCACTGGAGATATCAGGTATGATGAGGCCATGGGTTACCCCATGGTGCAGCAGTGGCG GGTCCGGAGCAACCTCTACCGTGTGAAGCTCAGCACCATCACCCTCGCAGCAGGCTTCACTAATGTTCTCAAGATTC TGACCAAGGAGAGCAGTCGGGAGGAGCTGCTGTCCTTCATCCAGCACTATGGCTCCCACTACATCGCAGAGGCCCTC TATGGCTCAGAGCTCACCTGCATCATCCACTTTCCCAGCAAGAAGGTCCAGCAGCAGCTGTGGCTCCAGTATCAGAA AGAGACCACAGAGCTGGGCAGCAAGAAGGAGCTCAAGTCCATGCCCTTCATCACCTACCTCTCAGGTTTGCTGACAG CCCAGATGCTGTCAGATGACCAGCTCATTTCAGGTGTGGAGATTCGCTGTGAGGAGAAGGGGCGCTGTCCATCTACC TGTCACCTTTGCCGCCGGCCAGGCAAGGAGCAGCTGAGCCCCACACCAGTGCTGCTGGAAATCAACCGTGTGGTGCC ACTTTATACCCTCATCCAAGACAATGGCACAAAGGAGGCCTTCAAGAGTGCACTGATGAGTTCCTACTGGTGCTCAG GGAAAGGGGATGTGATCGATGACTGGTGCAGGTGTGACCTCAGCGCCTTTGATGCCAATGGGCTCCCCAACTGCAGC CCCCTTCTGCAGCCGGTGCTGCGGCTGTCCCCAACAGTGGAGCCCTCCAGTACTGTGGTCTCCTTGGAGTGGGTGGA TGTTCAGCCAGCTATTGGGACCAAGGTCTCCGACTATATTCTGCAGCATAAGAAAGTGGATGAATACACAGACACTG ACCTGTACACAGGAGGATTCCTGAGTTTTGCTGATGACTTACTCTCTGGCCTGGGCACATCTTGTGTAGCAGCTGGT CGAAGCCATGGAGAGGTCCCTGAAGTCAGTATCTACTCAGTCATCTTCAAGTGTCTGGAGCCCGACGGTCTCTACAA GTTCACTCTGTATGCTGTGGATACACGAGGGAGGCACTCAGAGCTAAGCACGGTGACCCTGAGGACGGCCTGTCCAC TGGTAGATGACAACAAGGCAGAAGAAATAGCTGACAAGATCTACAATCTGTACAATGGGTACACAAGTGGAAAGGAG CAGCAGATGGCCTACAACACACTGATGGAGGTCTCAGCCTCGATGCTGTTCCGAGTCCAGCACCACTACAACTCTCA CTATGAAAAGTTTGGCGACTTCGTCTGGAGAAGTGAGGATGAGCTGGGGCCCAGGAAGGCCCACCTGATTCTACGGC GACTGGAGAGGGTGAGTAGCCACTGCTCCAGCCTCCTGCGGAGTGCCTACATCCAGAGCCGCGTGGAAACAGTGCCC TATCTTTTCTGCCGCAGCGAGGAGGTCCGGCCTGCAGGCATGGTGTGGTATAGCATCCTCAAGGACACCAAAATCAC GTGTGAGGAGAAGATGGTGTCAATGGCCCGAAACACGTACGGGGAGTCCAAGGGCCGG* AGCCAGTGTCCTGGGCTAAACACAAGAGTGCTGATTCCCACTGTAAGTTACAGTGAAGAACTTCTGCTATCTGAGGG CATGTGTTTTCATCTTCAAAAAAGGATGGACAGTCCCCATGAACCTTCCCTCTCCAACCACACAGGCCTTGCTTCTG GACATGCAGTGATAACTCTCTGTTTGCTGGATGAAGATCATGTTGGCTCTATGCACATTCAGATAACCTTCTACACC AGACACCCCTGGTGACTAGAGGGCCCTATTCTATAGTTCACCTAAA(SEQ ID NO.2)

Wherein, * is labeled as the site merged.

(2) amino acid sequence of fusion protein ASTN2-PAPPA

MAAAGARLSPGPGSGLRGRPRLCFHPGPPPLLPLLLLFLLLLPPPPLLAGATAAASREPDSPCRLKTVTVSTLPALR ESDIGWSGARAGAGAGTGAGAAAAAASPGSPGSAGTAAESRLLLFVRNELPGRIAVQDDLDNTELPFFTLEMSGTAA DISLVHWRQQWLENGTLYFHVSMSSSGQLAQATAPTLQEPSEIVEEQMHILHISVMGGLIALLLLLLVFTVALYAQR RWQKRRRIPQKSASTEATHEIHYIPSVLLGPQARESFRSSRLQTHNSVIGVPIRETPILDDYDCEEDEEPPRRANHV SREDEFGSQVTHTLDSLGHPGEEKVDFEKKAAAEATQETVESLMQKFKESFRANTPIEIGQLQPPLRSTSAGKRKRR SKSRGGISFGRAKGTSGSEADDETQLTFYTEQYRSRRRSKGLLKSPVNKTALTLIAVSSCILAMVCGSQMSCPLTVK VTLHVPEHFIADGSSFVVSEGSYLDISDWLNPAKLSLYYQINATSPWVRDLCGQRTTDACEQLCDPETGECSCHEGY APDPVHRHLCVRSDWGQSEGPWPYTTLERGYDLVTGEQAPEKILRSTFSLGQGLWLPVSKSFVVPPVELSINPLASC KTDVLVTEDPADVREEAMLSTYFETINDLLSSFGPVRDCSRNNGGCTRNFKCVSDRQVDSSGCVCPEELKPMKDGSG CYDHSKGIDCSDGFNGGCEQLCLQQTLPLPYDATSSTIFMFCGCVEEYKLAPDGKSCLMLSDVCEGPKCLKPDSKFN DTLFGEMLHGYNNRTQHVNQGQVFQMTFRENNFIKDFPQLADGLLVIPLPVEEQCRGVLSEPLPDLQLLTGDIRYDE AMGYPMVQQWRVRSNLYRVKLSTITLAAGFTNVLKILTKESSREELLSFIQHYGSHYIAEALYGSELTCIIHFPSKK VQQQLWLQYQKETTELGSKKELKSMPFITYLSGLLTAQMLSDDQLISGVEIRCEEKGRCPSTCHLCRRPGKEQLSPT PVLLEINRVVPLYTLIQDNGTKEAFKSALMSSYWCSGKGDVIDDWCRCDLSAFDANGLPNCSPLLQPVLRLSPTVEP SSTVVSLEWVDVQPAIGTKVSDYILQHKKVDEYTDTDLYTGGFLSFADDLLSGLGTSCVAAGRSHGEVPEVSIYSVI FKCLEPDGLYKFTLYAVDTRGRHSELSTVTLRTACPLVDDNKAEEIADKIYNLYNGYTSGKEQQMAYNTLMEVSASM LFRVQHHYNSHYEKFGDFVWRSEDELGPRKAHLILRRLERVSSHCSSLLRSAYIQSRVETVPYLFCRSEEVRPAGMV WYSILKDTKITCEEKMVSMARNTYGESKGR* SQCPGLNTRVLIPTVSYSEELLLSEGMCFHLQKRMDSPHEPSLSNHTGLASGHAVITLCLLDEDHVGSMHIQITFYT RHPW(SEQ ID NO.1)

Wherein, * is labeled as the site merged.

The building of 2 Antigen Expression Vectors of embodiment

1. the cloning primer that restriction enzyme site is introduced in design

F (Not I): 5 '-GTCAATGGCCCGAAACACG -3 ' (SEQ ID NO:3)

R (Xho I): 5 '-GGTTCATGGGGACTGTCCATC -3 ' (SEQ ID NO:4)

2. using Flag-A-P/ppyCAGIP plasmid as template (template schematic diagram is as shown in Figure 1), with F (NotI) and R (Xho I) primer carries out PCR and obtains 162bp target fragment, and the sequence of the mesh segment is shown in above-mentioned SEQID NO.2 with underscore；

3. using Not I and Xho I digestion PCR product, and glue recycling (electrophoresis result is as shown in Figure 2) is carried out, while using Not I and Xho I digestion pGEX-4T-1 plasmid (is purchased from Amersham Biosciences), and runs glue recycling 5Kb carrier segments；

4. connection insertion target fragment and carrier segments, 4 DEG C overnight；

5. converting connection product into bacillus coli DH 5 alpha；

6. choosing bacterium, small pumping digestion identification takes correct A-P/pGEX-4T-1 clone to be sequenced.

3 antigen purification of embodiment

1. converting the A-P/pGEX-4T-1 plasmid that embodiment 2 is prepared into BL21 competence；

2. choosing bacterium to 5ml YTA (peptone 16g/L；Yeast extract 10g/L；Sodium chloride 5g/L) it is medium and small shake overnight；

3.1: 30 inoculation 2L YTA shake 3h；30 DEG C of 0.2mM (GST) or 1mM (His) IPTG induce 3h；

Bacterium is received in 4.10000g, 3min centrifugation, and point 4 pipes dress is resuspended with 15ml PBS (GST) respectively, and be added at 1: 100

PMSF (final concentration 1mM)；

5. ultrasound, intensity: 10-12；Time: 8-10min；Interval: 2s；

6. 1%TritonX-100 is added after ultrasound, it is centrifuged 10000g, 10min, takes supernatant；

7. handling GST beads during centrifugation: washing 3 times, 10 times of volumes PBS, 500g, 5min；(storage: 80%) will centrifugation GST beads (every ml is in combination with 5-10mg albumen) is mixed supernatant of bacteria solution afterwards with treated, and 4 DEG C, rotation combines 2-4h；

8. washing: being washed 3 times with 10 times of volume PBS, each RT rotates 5-10min, centrifugation；

9. elution；Elution buffer: 0.1M Tris-cl, 0.2M Nacl, 61.5ng Glutathine, 0.1%

TritonX-100；4 DEG C of rotation 30min-1h, centrifugation；Elution 2-3 times；

10. running glue, coomassie brilliant blue staining is quantitative.

Wherein, GST is a label protein, it can be combined with glutathione.Therefore by the antigen polypeptide of the application with After GST fusion, paddy can be recycled by glutathione affine combination of the GST and immobilization on fusion protein on carrier The principle of the sweet peptide exchange elution of Guang carrys out purified fusion albumen.

4 immune rabbit of embodiment

The gst fusion protein antigen that embodiment 3 is prepared is injected into two adult male rats respectively, is carried out in two times It is immune, twice between interval 1-2 months or so, the gst fusion protein antigen being prepared in total with the embodiment of 5mg 3.

ELISA detection (ELISA result such as Fig. 3 institute is carried out to the potency of rabbit Serum Antibody after immune for the second time Show), when potency reaches 1:1000, rabbit bloodletting can be collected serum, be used for antibody purification.

5 antibody purification of embodiment (salting out method)

1. 5ml serum antibody-containing (being made by embodiment 4) is mixed in centrifuge tube in equal volume with 0.01M PBS, 10ml saturated ammonium sulfate solution is added thereto, is shaken up when being added dropwise, is subsequently placed in 2 to 8 degrees Celsius and stands overnight.

2. 8000r/min is centrifuged 15 to 20 minutes by the material to be separated in step 1 in being centrifuged in centrifuge, supernatant is removed.

3. the precipitating 2ml 0.01M PBS after being centrifuged in step 2 dissolves, it is then slowly added into 3ml saturated ammonium sulfate Solution shakes up when being added dropwise, and is subsequently placed in 2 to 8 degrees Celsius and stands 2 hours.

4. 8000r/min is centrifuged 15 to 20 minutes by the material to be separated in step 3 in being centrifuged in centrifuge, supernatant is removed.

5. the precipitating 1.65ml 0.01M PBS after being centrifuged in step 4 dissolves, it is then slowly added into 3.35ml saturation Ammonium sulfate shakes up when being added dropwise, and is subsequently placed in 2 to 8 degrees Celsius and stands 2 hours.

6. 8000r/min is centrifuged 15 to 20 minutes by the material to be separated in step 5 in being centrifuged in centrifuge, supernatant is removed.

7. the precipitating 1ml 0.01M PBS after being centrifuged in step 6 dissolves, it is transferred in 14000 bag filter of MD, uses 0.01M PBS dialyses, and dialysis 4 times or more, every time at least 1 hour or more.

It is saved 8. finally obtained antibody-solutions are dispensed, every pipe 500ul collects A-P overexpression group (A-P), A-P respectively The albumen of knockout group (shA-P) and wild-type cell (WT), WB detect the specificity of antibody.

As a result as shown in figure 4, detecting the A-P of higher level in A-P group, the A- of very low level is detected in shA-P group P detects medium level A-P in WT group, illustrates that the antibody can be with selectively targeted fusion protein A-P.

The application of 6 human fusion protein ASTN2-PAPPA polyclonal antibody of embodiment

(1) the human fusion protein A-P polyclonal antibody that high specific is prepared in the present invention is tested using Westernblot The differential expression (as shown in Figure 5) of tumour cell after detectable normal cell, tumour cell and medication.

(2) antibody provided by the invention can be applied to detect in the immunological testings such as IHC, Elisa in practical applications Situation of change of the human fusion protein A-P in normal tissue into cancerous tissue conversion process, to inquire into human fusion protein in tumour Meaning (as shown in Figure 6) in related disease.

(3) polyclonal antibody provided by the invention helps to inquire into fusion protein A-P in tumor disease occurrence and development process In mechanism of action, be more conducive to inhibit research of the drug to tumor inhibition effect in clinical application.The antibody of fusion protein A-P Application in terms of tumour correlative study and treatment, it is including but not limited to described above.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention And range.

SEQUENCE LISTING

<110>Zhang Hao

<120>a kind of fusion protein and polyclonal antibody and its application

<130> 2018

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1420

<212> PRT

<213>homo sapiens

<400> 1

Met Ala Ala Ala Gly Ala Arg Leu Ser Pro Gly Pro Gly Ser Gly Leu

1 5 10 15

Arg Gly Arg Pro Arg Leu Cys Phe His Pro Gly Pro Pro Pro Leu Leu

20 25 30

Pro Leu Leu Leu Leu Phe Leu Leu Leu Leu Pro Pro Pro Pro Leu Leu

35 40 45

Ala Gly Ala Thr Ala Ala Ala Ser Arg Glu Pro Asp Ser Pro Cys Arg

50 55 60

Leu Lys Thr Val Thr Val Ser Thr Leu Pro Ala Leu Arg Glu Ser Asp

65 70 75 80

Ile Gly Trp Ser Gly Ala Arg Ala Gly Ala Gly Ala Gly Thr Gly Ala

85 90 95

Gly Ala Ala Ala Ala Ala Ala Ser Pro Gly Ser Pro Gly Ser Ala Gly

100 105 110

Thr Ala Ala Glu Ser Arg Leu Leu Leu Phe Val Arg Asn Glu Leu Pro

115 120 125

Gly Arg Ile Ala Val Gln Asp Asp Leu Asp Asn Thr Glu Leu Pro Phe

130 135 140

Phe Thr Leu Glu Met Ser Gly Thr Ala Ala Asp Ile Ser Leu Val His

145 150 155 160

Trp Arg Gln Gln Trp Leu Glu Asn Gly Thr Leu Tyr Phe His Val Ser

165 170 175

Met Ser Ser Ser Gly Gln Leu Ala Gln Ala Thr Ala Pro Thr Leu Gln

180 185 190

Glu Pro Ser Glu Ile Val Glu Glu Gln Met His Ile Leu His Ile Ser

195 200 205

Val Met Gly Gly Leu Ile Ala Leu Leu Leu Leu Leu Leu Val Phe Thr

210 215 220

Val Ala Leu Tyr Ala Gln Arg Arg Trp Gln Lys Arg Arg Arg Ile Pro

225 230 235 240

Gln Lys Ser Ala Ser Thr Glu Ala Thr His Glu Ile His Tyr Ile Pro

245 250 255

Ser Val Leu Leu Gly Pro Gln Ala Arg Glu Ser Phe Arg Ser Ser Arg

260 265 270

Leu Gln Thr His Asn Ser Val Ile Gly Val Pro Ile Arg Glu Thr Pro

275 280 285

Ile Leu Asp Asp Tyr Asp Cys Glu Glu Asp Glu Glu Pro Pro Arg Arg

290 295 300

Ala Asn His Val Ser Arg Glu Asp Glu Phe Gly Ser Gln Val Thr His

305 310 315 320

Thr Leu Asp Ser Leu Gly His Pro Gly Glu Glu Lys Val Asp Phe Glu

325 330 335

Lys Lys Ala Ala Ala Glu Ala Thr Gln Glu Thr Val Glu Ser Leu Met

340 345 350

Gln Lys Phe Lys Glu Ser Phe Arg Ala Asn Thr Pro Ile Glu Ile Gly

355 360 365

Gln Leu Gln Pro Pro Leu Arg Ser Thr Ser Ala Gly Lys Arg Lys Arg

370 375 380

Arg Ser Lys Ser Arg Gly Gly Ile Ser Phe Gly Arg Ala Lys Gly Thr

385 390 395 400

Ser Gly Ser Glu Ala Asp Asp Glu Thr Gln Leu Thr Phe Tyr Thr Glu

405 410 415

Gln Tyr Arg Ser Arg Arg Arg Ser Lys Gly Leu Leu Lys Ser Pro Val

420 425 430

Asn Lys Thr Ala Leu Thr Leu Ile Ala Val Ser Ser Cys Ile Leu Ala

435 440 445

Met Val Cys Gly Ser Gln Met Ser Cys Pro Leu Thr Val Lys Val Thr

450 455 460

Leu His Val Pro Glu His Phe Ile Ala Asp Gly Ser Ser Phe Val Val

465 470 475 480

Ser Glu Gly Ser Tyr Leu Asp Ile Ser Asp Trp Leu Asn Pro Ala Lys

485 490 495

Leu Ser Leu Tyr Tyr Gln Ile Asn Ala Thr Ser Pro Trp Val Arg Asp

500 505 510

Leu Cys Gly Gln Arg Thr Thr Asp Ala Cys Glu Gln Leu Cys Asp Pro

515 520 525

Glu Thr Gly Glu Cys Ser Cys His Glu Gly Tyr Ala Pro Asp Pro Val

530 535 540

His Arg His Leu Cys Val Arg Ser Asp Trp Gly Gln Ser Glu Gly Pro

545 550 555 560

Trp Pro Tyr Thr Thr Leu Glu Arg Gly Tyr Asp Leu Val Thr Gly Glu

565 570 575

Gln Ala Pro Glu Lys Ile Leu Arg Ser Thr Phe Ser Leu Gly Gln Gly

580 585 590

Leu Trp Leu Pro Val Ser Lys Ser Phe Val Val Pro Pro Val Glu Leu

595 600 605

Ser Ile Asn Pro Leu Ala Ser Cys Lys Thr Asp Val Leu Val Thr Glu

610 615 620

Asp Pro Ala Asp Val Arg Glu Glu Ala Met Leu Ser Thr Tyr Phe Glu

625 630 635 640

Thr Ile Asn Asp Leu Leu Ser Ser Phe Gly Pro Val Arg Asp Cys Ser

645 650 655

Arg Asn Asn Gly Gly Cys Thr Arg Asn Phe Lys Cys Val Ser Asp Arg

660 665 670

Gln Val Asp Ser Ser Gly Cys Val Cys Pro Glu Glu Leu Lys Pro Met

675 680 685

Lys Asp Gly Ser Gly Cys Tyr Asp His Ser Lys Gly Ile Asp Cys Ser

690 695 700

Asp Gly Phe Asn Gly Gly Cys Glu Gln Leu Cys Leu Gln Gln Thr Leu

705 710 715 720

Pro Leu Pro Tyr Asp Ala Thr Ser Ser Thr Ile Phe Met Phe Cys Gly

725 730 735

Cys Val Glu Glu Tyr Lys Leu Ala Pro Asp Gly Lys Ser Cys Leu Met

740 745 750

Leu Ser Asp Val Cys Glu Gly Pro Lys Cys Leu Lys Pro Asp Ser Lys

755 760 765

Phe Asn Asp Thr Leu Phe Gly Glu Met Leu His Gly Tyr Asn Asn Arg

770 775 780

Thr Gln His Val Asn Gln Gly Gln Val Phe Gln Met Thr Phe Arg Glu

785 790 795 800

Asn Asn Phe Ile Lys Asp Phe Pro Gln Leu Ala Asp Gly Leu Leu Val

805 810 815

Ile Pro Leu Pro Val Glu Glu Gln Cys Arg Gly Val Leu Ser Glu Pro

820 825 830

Leu Pro Asp Leu Gln Leu Leu Thr Gly Asp Ile Arg Tyr Asp Glu Ala

835 840 845

Met Gly Tyr Pro Met Val Gln Gln Trp Arg Val Arg Ser Asn Leu Tyr

850 855 860

Arg Val Lys Leu Ser Thr Ile Thr Leu Ala Ala Gly Phe Thr Asn Val

865 870 875 880

Leu Lys Ile Leu Thr Lys Glu Ser Ser Arg Glu Glu Leu Leu Ser Phe

885 890 895

Ile Gln His Tyr Gly Ser His Tyr Ile Ala Glu Ala Leu Tyr Gly Ser

900 905 910

Glu Leu Thr Cys Ile Ile His Phe Pro Ser Lys Lys Val Gln Gln Gln

915 920 925

Leu Trp Leu Gln Tyr Gln Lys Glu Thr Thr Glu Leu Gly Ser Lys Lys

930 935 940

Glu Leu Lys Ser Met Pro Phe Ile Thr Tyr Leu Ser Gly Leu Leu Thr

945 950 955 960

Ala Gln Met Leu Ser Asp Asp Gln Leu Ile Ser Gly Val Glu Ile Arg

965 970 975

Cys Glu Glu Lys Gly Arg Cys Pro Ser Thr Cys His Leu Cys Arg Arg

980 985 990

Pro Gly Lys Glu Gln Leu Ser Pro Thr Pro Val Leu Leu Glu Ile Asn

995 1000 1005

Arg Val Val Pro Leu Tyr Thr Leu Ile Gln Asp Asn Gly Thr Lys

1010 1015 1020

Glu Ala Phe Lys Ser Ala Leu Met Ser Ser Tyr Trp Cys Ser Gly

1025 1030 1035

Lys Gly Asp Val Ile Asp Asp Trp Cys Arg Cys Asp Leu Ser Ala

1040 1045 1050

Phe Asp Ala Asn Gly Leu Pro Asn Cys Ser Pro Leu Leu Gln Pro

1055 1060 1065

Val Leu Arg Leu Ser Pro Thr Val Glu Pro Ser Ser Thr Val Val

1070 1075 1080

Ser Leu Glu Trp Val Asp Val Gln Pro Ala Ile Gly Thr Lys Val

1085 1090 1095

Ser Asp Tyr Ile Leu Gln His Lys Lys Val Asp Glu Tyr Thr Asp

1100 1105 1110

Thr Asp Leu Tyr Thr Gly Gly Phe Leu Ser Phe Ala Asp Asp Leu

1115 1120 1125

Leu Ser Gly Leu Gly Thr Ser Cys Val Ala Ala Gly Arg Ser His

1130 1135 1140

Gly Glu Val Pro Glu Val Ser Ile Tyr Ser Val Ile Phe Lys Cys

1145 1150 1155

Leu Glu Pro Asp Gly Leu Tyr Lys Phe Thr Leu Tyr Ala Val Asp

1160 1165 1170

Thr Arg Gly Arg His Ser Glu Leu Ser Thr Val Thr Leu Arg Thr

1175 1180 1185

Ala Cys Pro Leu Val Asp Asp Asn Lys Ala Glu Glu Ile Ala Asp

1190 1195 1200

Lys Ile Tyr Asn Leu Tyr Asn Gly Tyr Thr Ser Gly Lys Glu Gln

1205 1210 1215

Gln Met Ala Tyr Asn Thr Leu Met Glu Val Ser Ala Ser Met Leu

1220 1225 1230

Phe Arg Val Gln His His Tyr Asn Ser His Tyr Glu Lys Phe Gly

1235 1240 1245

Asp Phe Val Trp Arg Ser Glu Asp Glu Leu Gly Pro Arg Lys Ala

1250 1255 1260

His Leu Ile Leu Arg Arg Leu Glu Arg Val Ser Ser His Cys Ser

1265 1270 1275

Ser Leu Leu Arg Ser Ala Tyr Ile Gln Ser Arg Val Glu Thr Val

1280 1285 1290

Pro Tyr Leu Phe Cys Arg Ser Glu Glu Val Arg Pro Ala Gly Met

1295 1300 1305

Val Trp Tyr Ser Ile Leu Lys Asp Thr Lys Ile Thr Cys Glu Glu

1310 1315 1320

Lys Met Val Ser Met Ala Arg Asn Thr Tyr Gly Glu Ser Lys Gly

1325 1330 1335

Arg Ser Gln Cys Pro Gly Leu Asn Thr Arg Val Leu Ile Pro Thr

1340 1345 1350

Val Ser Tyr Ser Glu Glu Leu Leu Leu Ser Glu Gly Met Cys Phe

1355 1360 1365

His Leu Gln Lys Arg Met Asp Ser Pro His Glu Pro Ser Leu Ser

1370 1375 1380

Asn His Thr Gly Leu Ala Ser Gly His Ala Val Ile Thr Leu Cys

1385 1390 1395

Leu Leu Asp Glu Asp His Val Gly Ser Met His Ile Gln Ile Thr

1400 1405 1410

Phe Tyr Thr Arg His Pro Trp

1415 1420

<210> 2

<211> 4339

<212> DNA

<213>homo sapiens

<400> 2

gattgatcta ccatggacta caaagacgat gacgacaagg ggatcatggc cgccgccggc 60

gcccggctca gccccggccc cggctcgggg ctccgggggc ggccgaggct ctgcttccac 120

ccggggccgc cgccactgct gccgctgctg ctgctgttcc tgctcctgct gccgccgccg 180

ccgctgctgg ccggcgccac cgccgctgcc tcgcgggagc ccgacagccc gtgccggctg 240

aagaccgtca cggtgtccac actgcccgcc ctgcgggaga gcgacatcgg ctggagcggc 300

gcccgcgccg gggccggggc tgggaccggg gccggagccg ccgccgccgc cgcgtccccg 360

ggctctcctg gctctgccgg caccgccgcc gagtcgcgcc tcctgctctt tgtgcgtaac 420

gagctgccgg ggcgcatcgc ggtgcaggac gacctggaca acaccgagct gcccttcttc 480

accctggaga tgtctggcac agcggcggac atctcgctgg tgcactggag acagcagtgg 540

ctggagaatg gcaccttgta cttccacgtc tccatgagca gctccgggca gctggcccaa 600

gccaccgccc ccactctcca ggagccctcg gagattgttg aggagcagat gcacatcctc 660

cacatttctg tgatgggtgg cctcatcgcg ctgctgctgc tgctgctggt gttcaccgtg 720

gcgctgtacg cccagcgacg ttggcagaag cgtcgccgca tcccccagaa gagcgcaagc 780

acagaagcca ctcatgagat ccactacatc ccatctgtgc tgctgggtcc ccaggcgcgg 840

gagagcttcc gttcatcccg gctgcaaacc cacaattccg tcattggcgt gcccatccgg 900

gagactccca tcctggatga ctatgactgt gaggaggatg aggagccacc taggcgggcc 960

aaccatgtct cccgcgagga cgagtttggc agccaggtga cccacactct ggacagtctg 1020

ggacatccag gggaagagaa ggtggacttt gagaagaaag cagcagctga ggcgactcag 1080

gagacagtgg agtccctgat gcagaagttc aaggagagtt tccgcgctaa cacgcccatc 1140

gagatcggtc agctgcaacc acccctgcgc agcacatcgg cagggaagag gaagcggagg 1200

agcaagtctc gaggaggaat cagctttggg agagccaagg ggacgtcggg ctcagaggca 1260

gacgatgaaa ctcagctgac attctacacg gagcagtacc gcagtcgccg ccgcagcaaa 1320

ggtttgctga aaagcccagt gaacaagaca gccctgacac tgattgctgt gagttcctgc 1380

atcctggcca tggtgtgtgg cagccagatg tcttgtccac tcactgtgaa ggtgactctg 1440

catgtgcccg agcacttcat agcagatgga agcagcttcg tggtgagtga agggagctac 1500

ctggacatct ccgactggtt aaacccagcc aagctttccc tgtattacca gatcaatgcc 1560

acctccccat gggtgaggga cctctgtgga caaaggacga cagatgcctg tgagcagctc 1620

tgcgacccag aaaccggaga gtgcagctgt catgaaggct atgcccctga ccctgttcac 1680

agacacctgt gtgtgcgcag tgactgggga cagagtgaag gaccttggcc ctacacgaca 1740

cttgagaggg gctatgatct ggtgacaggg gagcaagccc ctgaaaagat tctcaggtct 1800

actttcagct tgggccaagg cctctggctt cctgtcagca aaagctttgt ggttccgcct 1860

gtggagctgt ccatcaaccc cctggccagc tgcaagaccg atgtgctcgt cacggaagac 1920

cctgcagatg tcagggaaga agcgatgctg tccacatact ttgaaaccat caatgacctg 1980

ctgtcttcct tcgggccagt tcgtgactgc tctcggaaca atgggggctg cactcgcaac 2040

ttcaagtgtg tgtctgaccg gcaggtggat tcctcgggat gtgtgtgccc tgaggagctg 2100

aaacccatga aggatggctc tggctgctac gaccactcca aaggcattga ctgctctgat 2160

ggctttaatg gcggctgtga gcagctgtgc ctgcagcaga cgctgcccct gccctacgat 2220

gccacttcga gcaccatctt catgttctgc ggttgcgtgg aggagtacaa actggctcct 2280

gatggaaaat cctgcttaat gctctcagat gtctgcgagg gccccaagtg cctcaaacct 2340

gactccaaat tcaatgatac cctctttgga gagatgctac atggttacaa caaccggacc 2400

cagcatgtga accaaggcca agtcttccag atgaccttta gggagaacaa cttcatcaag 2460

gactttcccc agctggccga tgggctgttg gtgatcccgc tgccggtgga ggagcagtgc 2520

cggggggtcc tctccgagcc ccttccggac ctccaactgc tcactggaga tatcaggtat 2580

gatgaggcca tgggttaccc catggtgcag cagtggcggg tccggagcaa cctctaccgt 2640

gtgaagctca gcaccatcac cctcgcagca ggcttcacta atgttctcaa gattctgacc 2700

aaggagagca gtcgggagga gctgctgtcc ttcatccagc actatggctc ccactacatc 2760

gcagaggccc tctatggctc agagctcacc tgcatcatcc actttcccag caagaaggtc 2820

cagcagcagc tgtggctcca gtatcagaaa gagaccacag agctgggcag caagaaggag 2880

ctcaagtcca tgcccttcat cacctacctc tcaggtttgc tgacagccca gatgctgtca 2940

gatgaccagc tcatttcagg tgtggagatt cgctgtgagg agaaggggcg ctgtccatct 3000

acctgtcacc tttgccgccg gccaggcaag gagcagctga gccccacacc agtgctgctg 3060

gaaatcaacc gtgtggtgcc actttatacc ctcatccaag acaatggcac aaaggaggcc 3120

ttcaagagtg cactgatgag ttcctactgg tgctcaggga aaggggatgt gatcgatgac 3180

tggtgcaggt gtgacctcag cgcctttgat gccaatgggc tccccaactg cagccccctt 3240

ctgcagccgg tgctgcggct gtccccaaca gtggagccct ccagtactgt ggtctccttg 3300

gagtgggtgg atgttcagcc agctattggg accaaggtct ccgactatat tctgcagcat 3360

aagaaagtgg atgaatacac agacactgac ctgtacacag gaggattcct gagttttgct 3420

gatgacttac tctctggcct gggcacatct tgtgtagcag ctggtcgaag ccatggagag 3480

gtccctgaag tcagtatcta ctcagtcatc ttcaagtgtc tggagcccga cggtctctac 3540

aagttcactc tgtatgctgt ggatacacga gggaggcact cagagctaag cacggtgacc 3600

ctgaggacgg cctgtccact ggtagatgac aacaaggcag aagaaatagc tgacaagatc 3660

tacaatctgt acaatgggta cacaagtgga aaggagcagc agatggccta caacacactg 3720

atggaggtct cagcctcgat gctgttccga gtccagcacc actacaactc tcactatgaa 3780

aagtttggcg acttcgtctg gagaagtgag gatgagctgg ggcccaggaa ggcccacctg 3840

attctacggc gactggagag ggtgagtagc cactgctcca gcctcctgcg gagtgcctac 3900

atccagagcc gcgtggaaac agtgccctat cttttctgcc gcagcgagga ggtccggcct 3960

gcaggcatgg tgtggtatag catcctcaag gacaccaaaa tcacgtgtga ggagaagatg 4020

gtgtcaatgg cccgaaacac gtacggggag tccaagggcc ggagccagtg tcctgggcta 4080

aacacaagag tgctgattcc cactgtaagt tacagtgaag aacttctgct atctgagggc 4140

atgtgttttc atcttcaaaa aaggatggac agtccccatg aaccttccct ctccaaccac 4200

acaggccttg cttctggaca tgcagtgata actctctgtt tgctggatga agatcatgtt 4260

ggctctatgc acattcagat aaccttctac accagacacc cctggtgact agagggccct 4320

attctatagt tcacctaaa 4339

<210> 3

<211> 19

<212> DNA

<213>artificial sequence

<400> 3

gtcaatggcc cgaaacacg 19

<210> 4

<211> 21

<212> DNA

<213>artificial sequence

<400> 4

ggttcatggg gactgtccat c 21

Claims

1. a kind of fusion protein, which is characterized in that the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1 or institute The coded sequence of fusion protein is stated as shown in SEQ ID NO.2.

2. a kind of polyclonal antibody, which is characterized in that fusion protein or the fusion of the antigen of the antibody for claim 1 An at least segment polypeptide in albumen.

3. antibody according to claim 2, which is characterized in that the polypeptide covers the position of fusion of the fusion protein.

4. the preparation method of antibody as claimed in claim 3, which comprises the steps of:

(1) Antigen Expression Vectors are constructed, wherein express melting for the fusion protein of the nucleotide sequence covering claim 1 of antigen Coincidence point or so at least nucleotide sequence of 12bp；

(3) the antigen immune rabbit after purification that is obtained with step (2) simultaneously collects serum, obtains the polyclonal antibody.

5. the preparation method according to claim 4, which is characterized in that further include step (4): using salting out method purification step (3) gained serum is to obtain polyclonal antibody.

6. the preparation method according to claim 4, which is characterized in that the carrier uses plasmid ppyCAGIP.

7. the preparation method according to claim 4, which is characterized in that the cell is BL21.

8. polyclonal antibody described in claim 2 or 3 is in the fusion protein of preparation detection claim 1 and its examination of table level Application in agent box.

9. polyclonal antibody described in claim 2 or 3 is in the drug of preparation treatment tumour or the fusion egg of research claim 1 Application in white.

10. a kind of drug for treating tumour, which is characterized in that the drug contains Anti-TNF-α described in claim 2 or 3 Body.