CN110950958A - anti-BRCA 2 monoclonal antibody and application thereof - Google Patents
anti-BRCA 2 monoclonal antibody and application thereof Download PDFInfo
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- CN110950958A CN110950958A CN201911087870.6A CN201911087870A CN110950958A CN 110950958 A CN110950958 A CN 110950958A CN 201911087870 A CN201911087870 A CN 201911087870A CN 110950958 A CN110950958 A CN 110950958A
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- 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
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- 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
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- G—PHYSICS
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- 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
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- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-BRCA 2 monoclonal antibody and application thereof. The invention uses the purified recombinant BRCA2 protein fragment as immunogen to immunize BALB/c mice, extracts spleen lymphocytes of the mice which are successfully immunized, fuses the lymphocytes with mouse myeloma cells SP2/0 through cell fusion technology, and obtains hybridoma cell strains which stably secrete monoclonal antibodies against BRCA2 after two rounds of subclone screening, thereby obtaining monoclonal antibodies against BRCA 2.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-BRCA 2 monoclonal antibody and application thereof.
Background
Ovarian cancer is the most lethal of gynecological malignancies, with more than 90% of ovarian cancers originating in epithelial tissues. Within the cell, PARP is primarily responsible for the repair of DNA Single Strand Breaks (SSBs); PARP inhibitors inhibit the repair of SSB, converting it into DNA Double Strand Breaks (DSB); the survival of cells that develop DSBs often depends on a robust Homologous Recombination (HR) repair pathway, carryingBRCAPatients with mutated ovarian cancer, due to defects in their HR pathway, are well candidates for PARP inhibitors. In recent years, researchers have found that PARP inhibitors have a certain inhibitory activity also in ovarian cancer, which is a disorder of HR repair due to other causes. The study therefore aimed to investigate patients with impaired HR repair in epithelial ovarian cancerThe ratio, the expression form and the possible mechanism of the PARP provide a prophase theoretical basis for the clinical medication of the late PARP inhibitor in the epithelial ovarian cancer.
The BRCA2 gene is inherited in an autosomal dominant manner and plays an important role in the development of ovarian cancer. There have been many studies to date which have demonstrated that mutations and polymorphisms in the BRCA2 gene have an effect on ovarian cancer susceptibility. The risk of ovarian cancer of BRCA2 gene mutation carriers is obviously increased. The early diagnosis has extremely important significance for treating ovarian cancer, and the effective early diagnosis can obviously improve the treatment efficiency and lead the prognosis condition of a patient to be obviously improved. A large proportion of ovarian cancer patients have been associated with tumor metastasis at the time of diagnosis. Statistically, about 90% of deaths from ovarian cancer are caused by distant metastasis. Therefore, how to effectively prevent ovarian cancer in early stage and discover early treatment in early stage becomes a hot project of the current global tumor research, and a feasible, rapid and accurate early detection method for ovarian cancer is established, which is very key to the clinical evaluation of genetic risk of ovarian cancer, treatment of ovarian cancer and improvement of prognosis of ovarian cancer.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an anti-BRCA 2 monoclonal antibody and application thereof.
The scheme of the invention is realized by the following technical means:
the invention utilizes the recombinant BRCA2 protein fragment as immunogen to immunize BALB/c mice, utilizes cell fusion technology to fuse spleen lymphocytes of mice successfully immunized with mouse myeloma cells, screens hybridoma cell strains capable of stably secreting anti-BRCA 2 monoclonal antibodies, produces a large amount of BRCA2 monoclonal antibodies, obtains high-purity anti-BRCA 2 monoclonal antibodies after purification, and has important significance for further research on biological diagnostic reagents, detection reagents and the like which take BRCA2 as a target spot.
In a first aspect, the present invention provides an anti-BRCA 2 monoclonal antibody, wherein the variable region amino acid sequence of the antibody heavy chain is as set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: 3, respectively.
In a specific embodiment, the invention also discloses a preparation method of the anti-BRCA 2 monoclonal antibody, the invention firstly uses BRCA2 protein to immunize a BALB/c mouse, then spleen lymphocytes of the mouse which is successfully immunized are fused with myeloma cells, a hybridoma cell line which specifically secretes the anti-BRCA 2 monoclonal antibody is obtained by screening positive clones, the cell is inoculated in the abdominal cavity of the BALB/c mouse sensitized by liquid paraffin in advance, the anti-BRCA 2 monoclonal antibody is produced in large quantity, and the high-purity anti-BRCA 2 monoclonal antibody is prepared by a protein purification mode.
The protein immunization is carried out for three times; the myeloma cell is SP2/0 myeloma cell.
The invention also provides application of the anti-BRCA 2 monoclonal antibody in preparation of an immunological detection tool.
The application of the anti-BRCA 2 monoclonal antibody in preparing a biological detection reagent for detecting BRCA 2.
Further, the application is the application for preparing a diagnostic reagent for immunologically detecting the BRCA2 protein antigen.
Compared with the prior art, the invention has the beneficial effects that:
the monoclonal antibody of BRCA2 has high specificity and capacity of being produced in great amount, and the titer of the antibody reaches 1 × 106The above. The monoclonal antibody of BRCA2 can specifically bind to BRCA2 protein, and reduces possible cross reaction. Can be used for immunological detection of cells, has wide market prospect and has important clinical significance for further developing detection and diagnostic reagents for detecting BRCA 2.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of fractions collected from the fragment of the recombinant protein BRCA 2;
FIG. 2 is a SDS-PAGE electrophoresis of fractions of purified anti-BRCA 2 monoclonal antibody;
FIG. 3 shows the results of the measurement of the immunotiter of the anti-BRCA 2 monoclonal antibody at different dilutions;
FIG. 4 is a graph showing the results of Western blot experiments performed by the anti-BRCA 2 monoclonal antibody on human ovarian cancer cell OVCAR5 and BRCA2 protein in human ovarian cancer cell SKOV 3.
Detailed Description
The present invention is further explained below with reference to the embodiments, and those skilled in the art can appropriately modify the process parameters to realize the invention based on the contents herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.
Example 1: preparation of BRCA2 monoclonal antibody
(1) Expression and purification of recombinant BRCA2 protein
Constructing plasmid pET-30a-BRCA2 containing BRCA2 protein, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown. The plasmid pET-30a-BRCA2 is transferred into escherichia coli BL21 by adopting a method of heat shock 90 s at 42 ℃, the expression of the target recombinant protein is induced, a nickel column (purchased from Thermo Fisher) is used for purifying the protein, and SDS-PAGE electrophoresis is used for detecting the expression of the recombinant protein. FIG. 1 is an SDS-PAGE electrophoresis of fractions collected from recombinant BRCA2 protein; as shown in FIG. 1, the BRCA2 protein was purified to obtain higher purity. Collecting protein, freeze-drying, and preparing for subsequent animal immunization.
(2) BALB/c mouse immunity and potency detection
BALB/c mice used in this example were purchased from the animal model research institute of Nanjing university. Mice were immunized by a conventional method (refer to "modern antibody technology and its applications" published by Beijing university Press and "guidance on antibody preparation and use" from scientific Press). The serum titer of the immunized mice is detected by an indirect ELISA method. Taking recombinant BRCA2 protein fragment, and adding 0.05 mol/L carbonate buffer solution with pH9.6 to BRCA2 eggDiluting to 1 mu g/mL, adding a 96-well enzyme label plate, coating each well with 100 mu L at 4 ℃ overnight, taking out the enzyme label plate coated with the overnight, washing for 3 times by TBS-T buffer solution, patting the enzyme label plate dry, and storing at 4 ℃ for later use. After 1 week of the second immunization, a proper amount of blood is collected from the tail vein of the mouse, serum is separated by centrifugation at 5000 g for 15min, the serum is diluted by a sample diluent (phosphate buffer containing 0.5% bovine serum albumin) according to a gradient of 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000, an ELISA plate to be detected is added to each hole at 100 muL, incubated for 1h at 37 ℃, washed for 3 times by TBS-T buffer, the ELISA plate is patted dry, HRP-labeled goat anti-mouse secondary antibody (purchased from Jackson Immuno Research) diluted at 1:5000 is added to each hole, and incubated for 30 min at 37 ℃. And (3) taking out the ELISA plate, washing the ELISA plate for 5 times by TBS-T buffer solution, adding 100 mu L of TMB substrate display solution into each hole, developing the ELISA plate for 10-15 min in a dark place at 37 ℃, then adding 50 mu L of stop solution, and reading the light absorption value under the wavelength of 450 nm of an ELISA reader. The selected serum titer reaches 1:105The mice are immunized for the third time;
(3) fusion and screening of hybridoma
Preparing feeder layer cells, preparing SP2/0 myeloma cells, and taking mouse spleen cells for cell fusion 3-4 days after the immunization is finished. The fused cells are plated (6 pieces of 96-well plates), hybridoma cells for producing monoclonal antibodies are screened by an indirect ELISA method, and hybridoma cell strains with the best antibody secretion are obtained by two rounds of subclone screening, wherein the numbers of the hybridoma cell strains are named as: 2-10-B7-G12.
(4) And (3) producing and purifying the anti-BRCA 2 monoclonal antibody.
Preparing mouse monoclonal antibody ascites by a conventional method, purifying the ascites antibody by a protein G method, verifying the purity of the antibody by SDS-PAGE electrophoresis, and obtaining an SDS-PAGE electrophoresis chart by collecting components of the purified anti-BRCA 2 monoclonal antibody in FIG. 2; as shown in FIG. 2, the 2-10-B7-G12 antibody was eluted with about 55kD IgG heavy chain and about 25kD IgG light chain to achieve higher purity.
Example 2: BRCA2 monoclonal antibody titer assay
BRCA2 protein was diluted to 1. mu.g/mL with 0.05 mol/L carbonate buffer, pH9.6, and coatedELISA plate, 100 μ L/hole, 4 ℃ overnight; the monoclonal antibodies were mixed at a ratio of 1:10 with a sample diluent (phosphate buffer containing 0.5% bovine serum albumin)3、1:104、1：105、1：106、1：107Diluting, 100 mu L/hole, and incubating for 1h at 37 ℃; taking out the ELISA plate, washing for 3 times by TBS-T, patting the ELISA plate dry, adding 100 μ L of HRP-labeled goat anti-mouse secondary antibody diluted by 1:5000 into each hole, and incubating for 30 min at 37 ℃; washing with TBS-T for 5 times, adding 100 μ L of TMB substrate display solution into each well, developing at 37 deg.C in dark for 10-15 min, adding 50 μ L of stop solution to terminate the reaction, and reading the absorbance at 450 nm wavelength of an enzyme labeling instrument, wherein FIG. 3 shows the detection results of the immune titer of the anti-BRCA 2 monoclonal antibodies with different dilutions; as shown in FIG. 3, the titers of the purified monoclonal antibodies all reached 1X 106。
Example 3: western blot detection of BRCA2 monoclonal antibody
Conventionally cultured human ovarian cancer cells OVCAR5 (purchased from the Kingming cell Bank of China) and human ovarian cancer cells SKOV3 (purchased from the Kingming cell Bank of China) were collected and subjected to Western blot detection using the anti-BRCA 2 monoclonal antibody prepared in example 1 according to a conventional method. As shown in fig. 4, the monoclonal antibody against BRCA2 obtained in the present invention can specifically recognize BRCA2 protein in OVCAR5 and SKOV3 cells, which indicates that the BRCA2 antibody prepared in the present invention has high specificity.
Example 4: sequencing of heavy and light chain variable region of BRCA2 monoclonal antibody
In order to solve the problems that the monoclonal cells are stored for a long time and positive clones are lost due to instability and pollution after multiple passages, the invention utilizes a molecular biology technology to amplify genes of a heavy chain variable region (mVH) and a light chain variable region (mVL) of a positive monoclonal cell strain by utilizing a Mouse Ig-PrimerSet kit (69831, Merck Millipore) and carry out sequence identification. Obtaining the amino acid sequence of the heavy chain variable region of the hybridoma cell as shown in SEQ ID NO: 2, namely: DVLLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRDRAKGYTEYSASVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARDDYGIRFTYWGQGTLVTVSA, respectively; the light chain variable region sequence amino acid sequence is shown as SEQ ID NO: 3, namely: DIVFTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWNSYPLTFGAGTKLELKR are provided.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Jiangsu
<120> anti-BRCA 2 monoclonal antibody and application thereof
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ccctataatt ctgaacctgc agaagaatct gaacataaaa acaacaatta cgaaccaaac 180
ctatttaaaa ctccacaaag gaaaccatct tataatcagc tggcttcaac tccaataata 240
ttcaaagagc aagggctgac tctgccgctg taccaatctc ctgtaaaaga attagataaa 300
ttcaaattag acttaggaag gaatgttccc aatagtagac ataaaagtct tcgcacagtg 360
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Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
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Gly Phe Ile Arg Asp Arg Ala Lys Gly Tyr Thr Glu Tyr Ser Ala Ser
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Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile Leu
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Tyr Leu Gln Met Asn Thr Leu Arg Thr Glu Asp Ser Ala Thr Tyr Tyr
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Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro Leu Thr
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Sequence listing
<110> university of Jiangsu
<120> anti-BRCA 2 monoclonal antibody and application thereof
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atgcctattg gatccaaaga gaggccaaca ttttttgaaa tttttaagac acgctgcaac 60
aaagcagatt taggaccaat aagtcttaat tggtttgaag aactttcttc agaagctcca 120
ccctataatt ctgaacctgc agaagaatct gaacataaaa acaacaatta cgaaccaaac 180
ctatttaaaa ctccacaaag gaaaccatct tataatcagc tggcttcaac tccaataata 240
ttcaaagagc aagggctgac tctgccgctg taccaatctc ctgtaaaaga attagataaa 300
ttcaaattag acttaggaag gaatgttccc aatagtagac ataaaagtct tcgcacagtg 360
aaaactaaaa tggatcaagc agat 384
<210>1
<211>119
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<213> human (Homo sapiens)
<400>1
Asp Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asp Arg Ala Lys Gly Tyr Thr Glu Tyr Ser Ala Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile Leu
65 70 75 80
Tyr Leu Gln Met Asn Thr Leu Arg Thr Glu Asp Ser Ala Thr Tyr Tyr
85 90 95
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Asp Ile Val Phe Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
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35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Tyr Pro Leu Thr
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Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
Claims (5)
1. An anti-BRCA 2 protein monoclonal antibody, wherein the antibody comprises a heavy chain variable region, the amino acid sequence of which is as shown in SEQ ID NO: 2, respectively.
2. The monoclonal antibody of claim 1, further comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 3, respectively.
3. Use of a monoclonal antibody according to any one of claims 1 or 2 for the preparation of an immunological detection means.
4. The use according to claim 3, for the preparation of a biological assay reagent for the detection of BRCA 2.
5. Use according to claim 3, for the preparation of a diagnostic reagent for the detection of BRCA2 antigen.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837492A (en) * | 1995-12-18 | 1998-11-17 | Myriad Genetics, Inc. | Chromosome 13-linked breast cancer susceptibility gene |
| WO1998055616A2 (en) * | 1997-06-06 | 1998-12-10 | Incyte Pharmaceuticals, Inc. | Brca2 locus-associated protein |
| CN108559775A (en) * | 2018-05-25 | 2018-09-21 | 北京泱深生物信息技术有限公司 | Dengue fever and dengue hemorrhagic fever diagnose diacritics object |
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2019
- 2019-11-08 CN CN201911087870.6A patent/CN110950958B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837492A (en) * | 1995-12-18 | 1998-11-17 | Myriad Genetics, Inc. | Chromosome 13-linked breast cancer susceptibility gene |
| WO1998055616A2 (en) * | 1997-06-06 | 1998-12-10 | Incyte Pharmaceuticals, Inc. | Brca2 locus-associated protein |
| CN108559775A (en) * | 2018-05-25 | 2018-09-21 | 北京泱深生物信息技术有限公司 | Dengue fever and dengue hemorrhagic fever diagnose diacritics object |
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| CN110950958B (en) | 2022-07-22 |
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