CN107188965A - Anti- DOG1 protein monoclonal antibodies and application thereof - Google Patents

Anti- DOG1 protein monoclonal antibodies and application thereof Download PDF

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Publication number
CN107188965A
CN107188965A CN201710535399.7A CN201710535399A CN107188965A CN 107188965 A CN107188965 A CN 107188965A CN 201710535399 A CN201710535399 A CN 201710535399A CN 107188965 A CN107188965 A CN 107188965A
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dog1
monoclonal antibody
oti1c6
albumen
kit
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何为无
马东晖
魏海涛
陈才伟
褚伯阳
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WUXI ORIGENE BIO-TECHNOLOGY CO LTD
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WUXI ORIGENE BIO-TECHNOLOGY CO LTD
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Publication of CN107188965A publication Critical patent/CN107188965A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Organic Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to biological technical field, a kind of hybridoma cell strain (deposit number is CGMCC No.13821), and the monoclonal antibody OTI1C6 that thus hybridoma cell strain is produced are disclosed.The application in being used to detect the immune detection instrument of DOG1 albumen is being prepared the invention further relates to monoclonal antibody OTI1C6, the immunohistochemical kit of the OTI1C6 containing monoclonal antibody, and applications of the monoclonal antibody OTI1C6 in the kit for marked tumor is prepared.Monoclonal antibody of the present invention can be combined with DOG1 protein-specifics, and with other intracellular albumen no cross reactions, significantly improve DOG1 protein immunizations detection specificity, accuracy and reliability.

Description

Anti- DOG1 protein monoclonal antibodies and application thereof
Technical field
The present invention relates to biological technical field, and in particular to can specific bond DOG1 albumen monoclonal antibody OTI1C6, Produce the cell line of the monoclonal antibody and apply the diagnostic method of the antibody and purposes.
Background technology
DOG1 (Discovered on gastrointestinal stromal tumors protein 1) is 2004 The specificity overexpression filtered out by West etc. using cDNA microarray technologies is in gastrointestinal stromal tumor (gene of (gastrointestinal stromal tumor, GISTs), alias ORAOV2, TMEM16A, FLJ10261 and ANO1, is positioned at human chromosome 11q13 sites, is 986 amino acid compositions, average molecular matter containing 26 extron code products Measure as 114kD.The albumen has 8 functional areas for wearing film, and research shows that it is probably the chloride channel protein of calcium ion regulatory.
Research confirms that DOG1 protein selectivity height is expressed in GISTs, and interstitial is mutated to Epithelial Gastric stromal tumors, PDGFR α Knurl, Extra-gastrointestinal Stromal Tumors, metastatic mesenchymoma and children's mesenchymoma have good sensitivity and specificity, and DOG1 exists Expression rate in GISTs is 90%~95%, be a kind of more sensitive, special mark and its expression be not rely on KIT or PDGFR α gene mutation situations.Clinically commonly use the table of albumen in immunohistochemistry (IHC) Pathological experiment detection tumour cell Up to situation, but the core of IHC experiments is the monoclonal antibody of binding proteins specific, and the quality of its performance directly decides whole The sensitivity and specificity of individual detection.Therefore, a kind of higher monoclonal for DOG1 albumen of binding specificity is developed to resist Body has great importance to IHC detection DOG1 expressions.
The content of the invention
In view of this, it is anti-it is an object of the invention to provide a kind of monoclonal of the higher DOG1 albumen of binding specificity Body, and its preparing the application in being used to detect the immune detection instrument of DOG1 albumen.
The invention provides a kind of hybridoma cell strain, China Committee for Culture Collection of Microorganisms is preserved in commonly micro- Bio-Centers (referred to as CGMCC), preservation date is on April 6th, 2017, and deposit number is CGMCC No.13821.
It is thin by above-mentioned hybridoma present invention also offers a kind of monoclonal antibody OTI1C6 of specific binding DOG1 albumen Born of the same parents' strain is produced.
The preparation method of monoclonal antibody of the present invention is as follows：
(1) structure of recombinant expression carrier：According to DOG1 ORF nucleotide sequences (DOG1 ORF nucleotide sequences such as SEQ Shown in ID NO.1,2961bp；DOG1 amino acid sequences are as shown in SEQ ID NO.2)
Primer PCR amplification DOG1 ORF 1bp are designed to 999bp bit sequences, gene both sides introduce restricted respectively Restriction enzyme site SgfI and MluI, insert expression vector pET23a-N-His, build DOG1 amino acid sequences the 1st to the 333rd The recombinant expression plasmid pET23a-rDOG1 of position；Upstream amplification primer sequence, SEQ ID NO.3: CACGCGATCGCATGAGGGTCAACGAGAAGT downstream amplification primer sequence SEQ ID NO.4: ACCGACGCGTCTAGGCGAAGTACAGGCCGATC
(2) expression and purification of DOG1 recombinant proteins：By DOG1 recombinant expression plasmid Transformed E .Coli cells, cracking centrifugation Supernatant is obtained, the purifying of affinity chromatography post obtains the DOG1 recombinant proteins of purifying；
(3) screening and preparation of monoclonal antibody：Balb/c mouse are immunized using the DOG1 recombinant proteins of above-mentioned purifying, taken Mouse spleen cells are merged with SP2/0 cells, and limiting dilution assay obtains monoclonal, and ELISA method screening positive hybridoma is thin Born of the same parents, acquisition can secrete the hybridoma cell strain of anti-DOG1 specific antibodies, be named as OTI1C6, hypotype is accredited as IgG2a；Pass through Serum free medium prepares antibody, and DOG1 monoclonal antibodies OTI1C6 is obtained by affinity column purifying.Pass through respectively Western Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.
Prepared present invention also offers monoclonal antibody OTI1C6 for detecting in the immune detection instrument of DOG1 albumen Application.
Specifically, the immune detection instrument is kit, chip or test paper.
In the particular embodiment, the invention provides a kind of immunologic combined detection reagent kit, including above-mentioned monoclonal is anti- Body OTI1C6, can detect the expression situation of DOG1 in histocyte.
Present invention also offers application of the said monoclonal antibody in the kit for marked tumor is prepared.Wherein institute State tumour and specifically refer to the propagation of tumour cell and the DOG1 closely related tumour of expression, including but not limited to Gastrointestinal Stromal Knurl, the carcinoma of the rectum.
Compared with prior art, the invention provides a kind of hybridoma cell strain, (deposit number is CGMCC No.13821 the monoclonal antibody OTI1C6 that), and thus hybridoma cell strain is produced.Present invention also offers monoclonal antibody OTI1C6 is preparing the application in being used to detect the immune detection instrument of DOG1 albumen, the immune group of the OTI1C6 containing monoclonal antibody Change kit, and applications of the monoclonal antibody OTI1C6 in the kit for marked tumor is prepared.List of the present invention Clonal antibody can be combined with DOG1 protein-specifics, DOG1 protein expression levels in true reflection tumour cell, can be applied to stomach The detection of Intestinal Stromal Tumors, carcinoma of the rectum DOG1 protein expression levels.
Preservation information
Classification And Nomenclature for the hybridoma cell strain OTI1C6 of preservation is：Anti-human (the Dog- of gastrointestinal stromal tumor albumen 1 of mouse 1) monoclonal hybridoma strain；
Depositary institution's full name：China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Depositary institution is referred to as：CGMCC；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；
Preservation date：On April 6th, 2017；
Deposit number：CGMCC No.13821.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 shows the design of the cloning site of embodiment 1 as schemed, and wherein shading part is ORF areas；
Fig. 2 show the formalin of embodiment 4 fix, the Human gastric stromal tumor immune groups of FFPE Change result figure (primary antibody is DOG1 monoclonal antibody OTI1C6)；
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all Belong to the scope of protection of the invention.
The structure of embodiment 1, DOG1 recombinant expression plasmids
With the plasmid RC229400 (2961bp containing DOG1ORF) obtained from bio tech ltd of Aureal Dongyuan County of the U.S. For template, design two primers and introduce restriction enzyme site SgfI and MluI respectively, be cloned into expression vector pET23a-N-His, build Vertical DOG1 recombinant expression plasmids.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, DOG1 recombinant proteins
1st, Transformed E .coli cells：Add DNA after 100ul competent cells are placed on ice to melt gently to mix, ice 42 DEG C of heat shock 90s after 30min are bathed, are then continued ice bath 1-2min.The fresh nonreactive LB cultures of 500ul are added in super-clean bench Base, takes appropriate bacterium solution to be spread evenly across on the flat board containing antibiotic, culture dish is inverted in into 37 after 37 DEG C of shaking tables are incubated 45min Overnight incubation in DEG C constant incubator.
2nd, cell lysis：Picking monoclonal is in fresh culture, 37 DEG C, 200rpm cultivates to OD values and reach 0.4~0.6 When add IPTG (final concentration 1mM) Fiber differentiation 7h.Thalline is collected by centrifugation, thalline then is resuspended with lysis buffer, ultrasound is broken Broken 20min centrifuges 20min after 12000rpm at 4 DEG C, collects supernatant.A small amount of supernatant protein is taken to make WB mirror with anti-His antibody It is fixed.
3rd, affinity chromatography post is purified：With buffer solution balance nickel post, by supernatant with loading after 0.45 μm of membrane filtration simultaneously Outflow is collected, is eluted with buffer solution and removes uncombined albumen, finally with the elution of the imidazoles containing various concentrations, received respectively SDS-PAGE identifications are carried out after collection, satisfactory elution albumen is merged and adds 10% glycerine, restructuring DOG1 eggs after purification Identified in vain with SDS-PAGE.
The preparation and screening of embodiment 3, DOG1 monoclonal antibodies
The DOG1 protein fragments of the purifying produced according to standard method with restructuring are used to (tie up tonneau in Beijing to Balb/c mouse Magnificent experimental animal Technology Co., Ltd.) it is immunized.Specific method is as follows：
1st, animal immune：Purified DOG1 antigens are emulsified with complete Freund's adjuvant, using subcutaneous or intraperitoneal injection side 6-8 week old Balb/c mouse are immunized in method, and for 50 μ g/ only, interval carries out being immunized for second immunizing dose after two weeks, with not exclusively not Family name's adjuvant emulsion, immunizing dose is 50 μ g/.It is immune to take tail blood to determine serum titer with ELISA method gradient dilution afterwards twice；Root Determine whether booster immunization according to result, choose antibody titer highest mouse and carry out cell fusion.
2nd, cell fusion：Myeloma cell uses the sp2/0 that Balb/c originates, and exponential phase is in during fusion；Take Immune mouse spleen, is made lymphocyte single cell suspension；Mouse spleen lymphocyte is with myeloma cell with 1:5-1:10 mixing, 37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, incomplete culture medium and remaining terminate liquid is added, centrifugation, which is abandoned, adds HAT after supernatant Culture medium, which suspends, to be mixed, and MC constant volumes are dispensed into 3.5cm culture dishes, are put in wet box to 50mL, are placed in 37 DEG C, 5%CO2It is permanent Cultivated in warm incubator.
3rd, screen and clone：Fusion selects cell clone in 7-10 days, and ELISA surveys are carried out using DOG1 purification of recombinant proteins Examination.Mark cell line number.Positive hole cell is carried out to determine ELISA values, picking within 5-6 days after limiting dilution, each limiting dilution The higher monoclonal hole of OD280 positive values carries out limiting dilution, until it is the positive that ELISA, which determines the complete hardened fruit of 96 orifice plates,.Picking The high monoclonal singling of positive value.Its correspondence fusion plate cell line is OTI1C6.
4th, the preparation and purification of ascites monoclonal antibody：The male Balb/c mouse peritoneal injections 0.5ml norphytanes of 10-12 week old, Every mouse is injected intraperitoneally with 1mL syringes after one week wash the monoclonal cell suspension being resuspended through PBS, cell consumption for 5 × 106/ only, make a call to 2 mouse per strain antibody.After collecting ascites after mouse ascites accumulation, centrifuging and taking supernatant, affinity chromatography carries out abdomen Water is purified, and corresponding post material is selected according to antibody subtype, and the monoclonal antibody that cell line OTI1C6 is produced is IgG2a, is entered using protein G Row purifying.Monoclonal antibody concentration mensuration after purification, WB are detected, dispense, frozen at -20 DEG C.
Embodiment 4, monoclonal antibody OTI1C6 detect for the SABC of primary antibody
(1), experimental method：
1st, take people's lymph node tissue and human tonsil's tissue block that formalin is fixed to carry out FFPE, use Finesse histotomes are cut into slices, and tissue thickness is 6 μm.
2nd, dewaxing and aquation：Analyze pure 3 × 10min of dimethylbenzene, absolute ethyl alcohol 3 × 10min, 95% ethanol 5min, 85% Ethanol 5min, 75% ethanol 5min, deionized water immersion 3min × 3 time
3rd, add antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker hot high pressure and repair 3min, treat height When pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, sample is taken out, then naturally cools to room temperature.Deionized water soaks 3min × 3 times.
4th, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is soaked 5min × 3 time.
5th, plus confining liquid (Normal Goat Serum of PBS+5% skimmed milk powers+5%), 37 DEG C of incubation 60min.
6th, confining liquid is removed, is not rinsed, DOG1 monoclonal antibodies (OTI1C6), thinner ratio is added：1:150, carried out using confining liquid Dilution.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, and 5min is washed every time.PBST (0.02%Tween-20) is washed 1 time, and 5min is washed every time.
7th, 1,37 DEG C of 2 (Catlog No.D37-15) reagent of Polink- kits is added dropwise to be incubated 10-20 minutes.Use PBS Washing 3 times, each 5min.2,37 DEG C of Polink-2 kits (Catlog No.D37-15) reagent is added dropwise to be incubated 10-20 minutes, Washed 3 times using PBS, each 5min.
8th, developed the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill water washing.
9th, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water is rinsed 3 times, is stored at room temperature 1min。
10th, it is dehydrated and transparent：75% ethanol 5min, 100% ethanol 5minx3 times, 85% ethanol 5min, 95% ethanol 5min, 100% 3 × 5min of ethanol；3 × 5min of dimethylbenzene, neutral gum mounting.
11st, microscopy, is shown in Fig. 2.
(2), experimental result：
From Fig. 2 results, the visible specific after birth dyeing in Human gastric stromal tumor.Knot Fruit is consistent with DOG1 positioning in the cell and tissue expression specificity, shows monoclonal antibody OTI1C6 available for immuning tissue The level of chemical detection DOG1 albumen.
SEQUENCE LISTING
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>Anti- DOG1 protein monoclonal antibodies and application thereof
<130> MP1502004
<160> 2
<170>PatentIn version 3.3
<210> 1
<211>2961
<212> DNA
<213>Artificial sequence
<400> 1
ORIGIN
1 ATGAGGGTCA ACGAGAAGTA CTCGACGCTC CCGGCCGAGG ACCGCAGCGT CCACATCATC
61 AACATCTGCG CCATCGAGGA CATCGGCTAC CTGCCGTCCG AGGGCACGCT GCTGAACTCC
121 TTATCTGTGG ACCCTGATGC CGAGTGCAAG TATGGCCTGT ACTTCAGGGA CGGCCGGCGC
181 AAGGTGGACT ACATCCTGGT GTACCATCAC AAGAGGCCCT CGGGCAACCG GACCCTGGTC
241 AGGAGGGTGC AGCACAGCGA CACCCCCTCT GGGGCTCGCA GCGTCAAGCA GGACCACCCC
301 CTGCCGGGCA AGGGGGCGTC GCTGGATGCA GGCTCGGGGG AGCCCCCGAT GGACTACCAC
361 GAGGATGACA AGCGCTTCCG CAGGGAGGAG TACGAGGGCA ACCTCCTGGA GGCGGGCCTG
421 GAGCTGGAGC GGGACGAGGA CACTAAAATC CACGGAGTCG GGTTTGTGAA AATCCATGCC
481 CCCTGGAACG TGCTGTGCAG AGAGGCCGAG TTTCTGAAAC TGAAGATGCC GACGAAGAAG
541 ATGTACCACA TTAATGAGAC CCGTGGCCTC CTGAAAAAAA TCAACTCTGT GCTCCAGAAA
601 ATCACAGATC CCATCCAGCC CAAAGTGGCT GAGCACAGGC CCCAGACCAT GAAGAGACTC
661 TCCTATCCCT TCTCCCGGGA GAAGCAGCAT CTATTTGACT TGTCTGATAA GGATTCCTTT
721 TTCGACAGCA AAACCCGGAG CACGATTGTC TATGAGATCT TGAAGAGAAC GACGTGTACA
781 AAGGCCAAGT ACAGCATGGG CATCACGAGC CTGCTGGCCA ATGGTGTGTA CGCGGCTGCA
841 TACCCACTGC ACGATGGAGA CTACAACGGT GAAAACGTCG AGTTCAACGA CAGAAAACTC
901 CTGTACGAAG AGTGGGCACG CTATGGAGTT TTCTATAAGT ACCAGCCCAT CGACCTGGTC
961 AGGAAGTATT TTGGGGAGAA GATCGGCCTG TACTTCGCCT GGCTGGGCGT GTACACCCAG
1021 ATGCTCATCC CTGCCTCCAT CGTGGGAATC ATTGTCTTCC TGTACGGATG CGCCACCATG
1081 GATGAAAACA TCCCCAGCAT GGAGATGTGT GACCAGAGAC ACAATATCAC CATGTGCCCG
1141 CTTTGCGACA AGACCTGCAG CTACTGGAAG ATGAGCTCAG CCTGCGCCAC GGCCCGCGCC
1201 AGCCACCTCT TCGACAACCC CGCCACGGTC TTCTTCTCTG TCTTCATGGC CCTCTGGGCT
1261 GCCACCTTCA TGGAGCACTG GAAGCGGAAA CAGATGCGAC TCAACTACCG CTGGGACCTC
1321 ACGGGCTTTG AAGAGGAAGA GGAGGCTGTC AAGGATCATC CTAGAGCTGA ATACGAAGCC
1381 AGAGTCTTGG AGAAGTCTCT GAAGAAAGAG TCCAGAAACA AAGAGAAGCG CCGGCATATT
1441 CCAGAGGAGT CAACAAACAA ATGGAAGCAG AGGGTTAAGA CAGCCATGGC GGGGGTGAAA
1501 TTGACTGACA AAGTGAAGCT GACATGGAGA GATCGGTTCC CAGCCTACCT CACTAACTTG
1561 GTCTCCATCA TCTTCATGAT TGCAGTGACG TTTGCCATCG TCCTCGGCGT CATCATCTAC
1621 AGAATCTCCA TGGCCGCCGC CTTGGCCATG AACTCCTCCC CCTCCGTGCG GTCCAACATC
1681 CGGGTCACAG TCACAGCCAC CGCAGTCATC ATCAACCTAG TGGTCATCAT CCTCCTGGAC
1741 GAGGTGTATG GCTGCATAGC CCGATGGCTC ACCAAGATCG AGGTCCCAAA GACGGAGAAA
1801 AGCTTTGAGG AGAGGCTGAT CTTCAAGGCT TTCCTGCTGA AGTTTGTGAA TTCCTACACC
1861 CCCATCTTTT ACGTGGCGTT CTTCAAAGGC CGGTTTGTTG GACGCCCGGG CGACTACGTG
1921 TACATTTTCC GTTCCTTCCG AATGGAAGAG TGTGCGCCAG GGGGCTGCCT GATGGAGCTA
1981 TGCATCCAGC TCAGCATCAT CATGCTGGGG AAACAGCTGA TCCAGAACAA CCTGTTCGAG
2041 ATCGGCATCC CGAAGATGAA GAAGCTCATC CGCTACCTGA AGCTGAAGCA GCAGAGCCCC
2101 CCTGACCACG AGGAGTGTGT GAAGAGGAAA CAGCGGTACG AGGTGGATTA CAACCTGGAG
2161 CCCTTCGCGG GCCTCACCCC AGAGTACATG GAAATGATCA TCCAGTTTGG CTTCGTCACC
2221 CTGTTTGTCG CCTCCTTCCC CCTGGCCCCA CTGTTTGCGC TGCTGAACAA CATCATCGAG
2281 ATCCGCCTGG ACGCCAAAAA GTTTGTCACT GAGCTCCGAA GGCCGGTAGC TGTCAGAGCC
2341 AAAGACATCG GAATCTGGTA CAATATCCTC AGAGGCATTG GGAAGCTTGC TGTCATCATC
2401 AATGCCTTCG TGATCTCCTT CACGTCTGAC TTCATCCCGC GCCTGGTGTA CCTCTACATG
2461 TACAGTAAGA ACGGGACCAT GCACGGCTTC GTCAACCACA CCCTCTCCTC CTTCAACGTC
2521 AGTGACTTCC AGAACGGCAC GGCCCCCAAT GACCCCCTGG ACCTGGGCTA CGAGGTGCAG
2581 ATCTGCAGGT ATAAAGACTA CCGAGAGCCG CCGTGGTCGG AAAACAAGTA CGACATCTCC
2641 AAGGACTTCT GGGCCGTCCT GGCAGCCCGG CTGGCGTTTG TCATCGTCTT CCAGAACCTG
2701 GTCATGTTCA TGAGCGACTT TGTGGACTGG GTCATCCCGG ACATCCCCAA GGACATCAGC
2761 CAGCAGATCC ACAAGGAGAA GGTGCTCATG GTGGAGCTGT TCATGCGGGA GGAGCAAGAC
2821 AAGCAGCAGC TGCTGGAAAC CTGGATGGAG AAGGAGCGGC AGAAGGACGA GCCGCCGTGC
2881 AACCACCACA ACACCAAAGC CTGCCCAGAC AGCCTCGGCA GCCCAGCCCC CAGCCATGCC
2941 TACCACGGGG GCGTCCTGTA G
//
<210> 2
<211>986
<212> PRT
<213>Artificial sequence
<400> 2
ORIGIN
1 MRVNEKYSTL PAEDRSVHII NICAIEDIGY LPSEGTLLNS LSVDPDAECK YGLYFRDGRR
61 KVDYILVYHH KRPSGNRTLV RRVQHSDTPS GARSVKQDHP LPGKGASLDA GSGEPPMDYH
121 EDDKRFRREE YEGNLLEAGL ELERDEDTKI HGVGFVKIHA PWNVLCREAE FLKLKMPTKK
181 MYHINETRGL LKKINSVLQK ITDPIQPKVA EHRPQTMKRL SYPFSREKQH LFDLSDKDSF
241 FDSKTRSTIV YEILKRTTCT KAKYSMGITS LLANGVYAAA YPLHDGDYNG ENVEFNDRKL
301 LYEEWARYGV FYKYQPIDLV RKYFGEKIGL YFAWLGVYTQ MLIPASIVGI IVFLYGCATM
361 DENIPSMEMC DQRHNITMCP LCDKTCSYWK MSSACATARA SHLFDNPATV FFSVFMALWA
421 ATFMEHWKRK QMRLNYRWDL TGFEEEEEAV KDHPRAEYEA RVLEKSLKKE SRNKEKRRHI
481 PEESTNKWKQ RVKTAMAGVK LTDKVKLTWR DRFPAYLTNL VSIIFMIAVT FAIVLGVIIY
541 RISMAAALAM NSSPSVRSNI RVTVTATAVI INLVVIILLD EVYGCIARWL TKIEVPKTEK
601 SFEERLIFKA FLLKFVNSYT PIFYVAFFKG RFVGRPGDYV YIFRSFRMEE CAPGGCLMEL
661 CIQLSIIMLG KQLIQNNLFE IGIPKMKKLI RYLKLKQQSP PDHEECVKRK QRYEVDYNLE
721 PFAGLTPEYM EMIIQFGFVT LFVASFPLAP LFALLNNIIE IRLDAKKFVT ELRRPVAVRA
781 KDIGIWYNIL RGIGKLAVII NAFVISFTSD FIPRLVYLYM YSKNGTMHGF VNHTLSSFNV
841 SDFQNGTAPN DPLDLGYEVQ ICRYKDYREP PWSENKYDIS KDFWAVLAAR LAFVIVFQNL
901 VMFMSDFVDW VIPDIPKDIS QQIHKEKVLM VELFMREEQD KQQLLETWME KERQKDEPPC
961 NHHNTKACPD SLGSPAPSHA YHGGVL

Claims (7)

1. a kind of monoclonal antibody OTI1C6 of specific binding DOG1 albumen, is the miscellaneous of CGMCC No.13821 by deposit number Tumor cell strain is handed over to produce.
2. a kind of hybridoma cell strain, its deposit number is CGMCC No.13821.
3. monoclonal antibody as claimed in claim 1 is being prepared for detecting answering in the immune detection instrument of DOG1 albumen With.
4. application according to claim 3, the immune detection instrument is kit, chip or test paper.
5. a kind of immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.
6. application of the monoclonal antibody as claimed in claim 1 in the kit for tagged tissue cell is prepared.
7. application according to claim 6, the histocyte is gastrointestinal stromal tumor, the carcinoma of the rectum.
CN201710535399.7A 2017-07-06 2017-07-06 Anti- DOG1 protein monoclonal antibodies and application thereof Pending CN107188965A (en)

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CN113045652A (en) * 2021-04-09 2021-06-29 福州迈新生物技术开发有限公司 anti-DOG 1 protein monoclonal antibody and cell strain, preparation method and application thereof
CN113754764A (en) * 2021-08-20 2021-12-07 四川大学华西医院 anti-DOG-1 antibodies, conjugates, pharmaceutical compositions, pharmaceutical formulations and uses thereof
CN113801231A (en) * 2021-08-20 2021-12-17 四川大学华西医院 anti-DOG-1 antibodies or antigen-binding fragments thereof and uses thereof

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CN105131114A (en) * 2015-10-26 2015-12-09 无锡傲锐东源生物科技有限公司 P16 protein resistant monoclonal antibody hybridoma cell, p16 protein resistant monoclonal antibody generated by same and application thereof
CN106854245A (en) * 2016-12-28 2017-06-16 无锡傲锐东源生物科技有限公司 Protein monoclonal antibody of AntiCD3 McAb 0 and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113045652A (en) * 2021-04-09 2021-06-29 福州迈新生物技术开发有限公司 anti-DOG 1 protein monoclonal antibody and cell strain, preparation method and application thereof
CN113045652B (en) * 2021-04-09 2022-04-12 福州迈新生物技术开发有限公司 anti-DOG 1 protein monoclonal antibody and cell strain, preparation method and application thereof
CN113754764A (en) * 2021-08-20 2021-12-07 四川大学华西医院 anti-DOG-1 antibodies, conjugates, pharmaceutical compositions, pharmaceutical formulations and uses thereof
CN113801231A (en) * 2021-08-20 2021-12-17 四川大学华西医院 anti-DOG-1 antibodies or antigen-binding fragments thereof and uses thereof

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Application publication date: 20170922