CN117024563B - 分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 - Google Patents
分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 Download PDFInfo
- Publication number
- CN117024563B CN117024563B CN202310718836.4A CN202310718836A CN117024563B CN 117024563 B CN117024563 B CN 117024563B CN 202310718836 A CN202310718836 A CN 202310718836A CN 117024563 B CN117024563 B CN 117024563B
- Authority
- CN
- China
- Prior art keywords
- gamma
- kit
- interferon
- hybridoma cell
- experimental
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010074328 Interferon-gamma Proteins 0.000 title claims abstract description 65
- 241000282693 Cercopithecidae Species 0.000 title claims abstract description 42
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 37
- 102000008070 Interferon-gamma Human genes 0.000 title claims abstract description 30
- 229940044627 gamma-interferon Drugs 0.000 title claims abstract description 28
- 230000003248 secreting effect Effects 0.000 title claims abstract description 7
- 238000002965 ELISA Methods 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 17
- 238000004321 preservation Methods 0.000 claims description 8
- 238000003018 immunoassay Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 claims description 2
- 238000003127 radioimmunoassay Methods 0.000 claims description 2
- 238000003118 sandwich ELISA Methods 0.000 claims description 2
- 102100037850 Interferon gamma Human genes 0.000 abstract description 34
- 238000000034 method Methods 0.000 abstract description 12
- 238000002360 preparation method Methods 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000010171 animal model Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 206010003445 Ascites Diseases 0.000 description 15
- 230000003053 immunization Effects 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 238000002649 immunization Methods 0.000 description 13
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 238000011068 loading method Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 102000014150 Interferons Human genes 0.000 description 6
- 108010050904 Interferons Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000002250 absorbent Substances 0.000 description 5
- 230000002745 absorbent Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 229940079322 interferon Drugs 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 230000003187 abdominal effect Effects 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012474 protein marker Substances 0.000 description 3
- 239000012264 purified product Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010014726 Interferon Type I Proteins 0.000 description 2
- 102000002227 Interferon Type I Human genes 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101100454807 Caenorhabditis elegans lgg-1 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102100026688 Interferon epsilon Human genes 0.000 description 1
- 101710147309 Interferon epsilon Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002534 Polyethylene Glycol 1450 Polymers 0.000 description 1
- 208000010362 Protozoan Infections Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 108010018844 interferon type III Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
-
- 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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/249—Interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/57—IFN-gamma
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及一种分泌实验猴γ‑干扰素单克隆抗体的杂交瘤细胞及其应用,通过体外培养该杂交瘤细胞,可以获得大量实验猴γ‑干扰素单克隆抗体,具有制备方法简单、抗体滴度较高、易于纯化等优点;本发明提供的杂交瘤细胞及通过其制备的实验猴γ‑干扰素单克隆抗体,为开发特异性强、灵敏度高的猴IFN‑γELISA检测试剂盒等相关检测产品提供关键试剂,从而为实验动物猴的IFN‑γ检测提供有力的技术支持。
Description
技术领域
本发明涉及免疫学技术领域,具体涉及一种分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用。
背景技术
干扰素(IFN)是在特定的诱生剂作用下由细胞分泌的一类具有抗病毒、抗肿瘤和免疫调节功能等生物学活性的糖蛋白,分子量约为20~100kDa。根据基因序列和受体的不同,可以将IFN分为Ⅰ、Ⅱ和Ⅲ型3种类型。Ⅰ型主要包括IFN-α、β,还有后来发现的IFN-ε、ψ、κ、δ、τ、ξ等,是由成纤维细胞和上皮细胞被抗原刺激后分泌产生的。Ⅱ型干扰素,是由抗原、有丝分裂素等活化免疫细胞分泌产生的,因此又称为免疫干扰素。Ⅲ型干扰素是新发现的一类细胞因子,与Ⅰ型干扰素关系密切,称为IFN-λ。Ⅰ型干扰素主要表现为抗病毒、抗肿瘤的生物学活性;而Ⅱ型干扰素主要表现为免疫调节的生物学活性。
γ-干扰素(IFN-γ)是可溶性二聚体细胞因子,是II型干扰素的唯一成员,主要由CD4+Th1细胞、CD8+T细胞及NK细胞分泌,在对抗病毒、某些细菌和原生动物感染的固有免疫和适应性免疫具有重要作用。由于其在免疫反应的重要作用,近年来,在牛、猪、山羊、鸡等动物的γ-干扰素基因相继被成功克隆和表达,但目前国内关于猴干扰素的研究却鲜有报道。现有的实验猴IFN-γ的ELISA检测中,使用的捕获抗体大部分来源于国外,这给实验猴的相关检测带来了很大的成本和效率问题。
发明内容
本发明的目的在于提供一种分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用,通过体外培养该杂交瘤细胞,可以获得大量实验猴γ-干扰素单克隆抗体。
为此,本发明的第一方面提供一种重组抗原,其氨基酸序列如SEQ ID NO:1所示。
本发明的第二方面,提供一种生物材料,其为(A1)、(A2)、(A3)中的任意一种:
(A1)核酸分子,其编码本发明第一方面所述的重组抗原;
(A2)载体,其包括(A1)中所述的核酸分子;
(A3)宿主细胞,其表达本发明第一方面所述的重组抗原,和/或含有(A1)中所述的核酸分子,和/或含有(A2)中所述的载体。
在一些实施方式中,所述核酸分子包括SEQ ID NO:2所示的核苷酸序列。
在一些实施方式中,所述宿主细胞为原核细胞或真核细胞。
在一些实施方式中,所述宿主细胞选自酿酒酵母、毕赤酵母、链霉菌、枯草芽孢杆菌或大肠杆菌中的至少一种。
本发明的第三方面,提供所述重组抗原的制备方法,其包括在适于表达所述重组抗原的条件下培养本发明第二方面的(A3)中所述的宿主细胞,对培养得到的产物进行分离纯化,即制备得到所述重组抗原。
本发明的第四方面,提供一种分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞株,命名为杂交瘤细胞株6F7F3D3A11A4C4(Hybridoma cell line6F7F3D3A11A4C4),保藏编号为CCTCC NO:C202358,保藏日期为2023年4月5日,保藏于中国典型培养物保藏中心,保藏地址中国武汉。
本发明的第五方面,提供一种实验猴γ-干扰素单克隆抗体,其由本发明第四方面所述的杂交瘤细胞株或其传代细胞株分泌产生。
根据本发明的技术方案,所述实验猴γ-干扰素单克隆抗体经鉴定为lgG1型。
本发明的第六方面,提供本发明第四方面所述的杂交瘤细胞株或本发明第五方面所述的实验猴γ-干扰素单克隆抗体在制备检测实验猴γ-干扰素试剂方面的应用。
本发明的第七方面,提供一种实验猴γ-干扰素的检测产品;其包括本发明第五方面所述的实验猴γ-干扰素单克隆抗体。
在一些实施方式中,所述实验猴γ-干扰素的检测产品为试剂、试纸条或试剂盒的至少一种。
在一些实施方式中,所述试剂盒包括选自下组的至少一种:胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒、微流体芯片试剂盒、荧光免疫试剂盒中。
在一些实施方式中,所述试剂盒包括选自下组的至少一种:双抗体夹心ELISA试剂盒、竞争抑制ELISA检测试剂盒。
与现有技术相比,本发明的技术方案具有以下有益效果:
(1)本发明筛选了实验猴IFN-γ蛋白上的特异性序列片段,并制备成重组抗原,通过应用该重组抗原对小鼠进行免疫,取得了良好的免疫效果,并经进一步筛选后制备得到杂交瘤细胞。该杂交瘤细胞通过体外细胞培养技术或体内腹水制备技术,即可获得大量单克隆抗体。采用该杂交瘤细胞制备猴γ-干扰素单克隆抗体,具有制备方法简单、抗体滴度较高、易于纯化等优点。
(2)本发明提供的杂交瘤细胞及通过其制备的实验猴γ-干扰素单克隆抗体,为开发特异性强、灵敏度高的猴IFN-γELISA检测试剂盒等相关检测产品提供关键试剂,从而为实验动物猴的IFN-γ检测提供有力的技术支持。
(3)现有的实验猴IFN-γ的ELISA检测中,使用的捕获抗体大部分来源于国外,这给实验猴的相关检测带来了很大的成本和效率问题,而本发明提供的杂交瘤细胞及其制备的抗体,不仅可以在很大程度上节省实验成本,还可提高实验过程中的效率,同时,还可以保证实验的特异性及灵敏性。
附图说明
通过阅读下文优选实施方式的详细描述,各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的,而并不认为是对本发明的限制。在附图中:
图1:纯化后的重组IFN-γ蛋白的SDS-PAGE图;M,蛋白质marker;1,纯化后的重组IFN-γ蛋白;
图2:腹水及纯化后抗体的SDS-PAGE图;M,蛋白质marker;1,A组腹水;2,B组腹水;3,C组腹水;4,A1抗体,上样量1μg;5,A1抗体,上样量2μg;
图3:A1抗体的WesternBlot图;M,蛋白质marker;1,阴性对照;2,A1抗体,加二抗;3,A1抗体,不加二抗;4,RD标准品,上样量16ng/mL;5,RD标准品,上样量32ng/mL。
具体实施方式
下面将更详细地描述本公开的示例性实施方式。应当理解,可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反,提供这些实施方式是为了能够更透彻地理解本公开,并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1重组IFN-γ蛋白
(1)实验猴伽马干扰素重组表达质粒的构建
根据实验猴伽马干扰素蛋白序列(NP_001028077),选取24aa-165aa片段作为抗原(氨基酸序列如SEQ ID NO:1所示),将该蛋白序列相应的核酸序列按照大肠杆菌密码子的偏好性进行优化(核苷酸序列如SEQ ID NO:2所示)。人工合成优化后的核酸序列同时在羧基端加6×His标签序列及终止密码子,并拼接至pET28a载体,构建重组表达质粒pET28a-IFN-γ。
SEQ ID NO:1
SEQ ID NO:2
将重组表达质粒pET28a-IFN-γ转化至DH5α感受态细胞,在含有50μg/mL卡那霉素的LB固体培养基,37℃培养过夜。挑取单菌落接种于3mL含有50μg/mL卡那霉素的LB液体培养基,在恒温振荡培养箱37℃、180rpm培养过夜。用高纯度质粒小提试剂盒制备质粒,将质粒送至测序公司测序,测序结果证明与理论预期一致,在-20℃保存备用。
(2)重组蛋白的表达
将重组表达质粒pET28a-IFN-γ转化至BL21(DE3)感受态细胞,在含有50μg/mL卡那霉素的LB固体培养基,37℃培养过夜。挑取单菌落接种于3mL含有50μg/mL卡那霉素的LB液体培养基,在恒温振荡培养箱37℃、180rpm培养过夜,再转接至1200ml含有50μg/mL卡那霉素的LB液体培养基,37℃、180rpm振荡培养。当OD600到0.8左右时加入终浓度0.5mM异丙基硫代半乳糖苷(IPTG),诱导培养5h;诱导培养结束后,在4℃,4000rpm离心10min收集菌体。
(3)重组蛋白的纯化
将菌体用缓冲液重悬。重悬液在冰水浴300w超声破碎细胞,4℃、4000rpm离心10min取上清。上清用0.45μm滤膜过滤后,经HisTrap HP纯化,得到纯化的重组蛋白IFN-γ,暂时存于4℃。
(4)重组蛋白的透析与浓缩
将步骤(3)得到的纯化产物加入到已用蒸馏水充分冲洗后的3.5kD Spectra透析袋,在4℃透析至透析缓冲液,纯化产物与透析液体积比为1:100,换液3次,共透析24h。透析后的纯化产物用超滤管进行浓缩。浓缩后产物用BCA蛋白浓度测定试剂盒测定蛋白浓度。将浓缩后产物分装保存于-80℃。结果显示:实验猴伽马干扰素重组蛋白在大肠杆菌中高效表达,1200mL培养液纯化后获得重组IFN-γ蛋白10mg,产率8.3mg/L。
(5)重组蛋白的SDS-PAGE鉴定
取15μL步骤(4)制备得到的重组蛋白,加入2×Loading Buffer 15μL,混合后煮沸5min,瞬离后取上清20μL上样到10% SDS-PAGE。用Bio-Rad电泳仪,110V电泳约1h。凝胶用考马斯亮蓝染色液浸没并在水平摇床上低速转动,室温染色约2h;去除染色液,加入脱色液浸没凝胶并在水平摇床上低速转动,约30min更换一次新的脱色液,脱色至条带清晰且背景无蓝色,脱色后的凝胶在凝胶成像系统中拍照记录,如图1所示,图1中1栏下面有1条较粗的深色条带,说明该方法获得了蛋白且蛋白与理论值相近。
实施例2双抗夹心法ELISA鉴定
取实施例1制备得到的重组IFN-γ蛋白,用1%BSA稀释至16ng/ml,4倍比稀释至0.25ng/ml,100μL/孔加入到包被有IFN-γ捕获蛋白的微孔板中,室温孵育2h;用0.05%PBST以350μL/孔洗涤3次,并在吸水纸上倒扣拍干;加入稀释好的生物素化抗体,100μL/孔,室温孵育2h;用0.05%PBST以350μL/孔洗涤3次,并在吸水纸上倒扣拍干;加入稀释好的酶标链霉亲和素,100μL/孔,室温孵育20min;用0.05%PBST以350μL/孔洗涤3次,并在吸水纸上倒扣拍干;加入显色试剂100μL/孔,室温孵育30min;加入终止试剂100μL/孔,在酶标仪上读数OD450和OD630,OD450-OD620的数值作为结果,结果如表1所示。测试结果表明,商业IFN-γ捕获蛋白可以与本发明提供的重组IFN-γ蛋白产生特异性反应,重组IFN-γ蛋白可用于后续实验。
表1重组IFN-γ蛋白双抗夹心法鉴定结果
实施例3小鼠免疫
取实施例1制备得到的重组IFN-γ蛋白,用缓冲液稀释成浓度为600μg/mL的重组蛋白备用。免疫7周龄雌性ICR小鼠5只,首次免疫:按照50μg/只的蛋白量,将重组蛋白和完全佛氏佐剂按体积比1:1混合,充分乳化后,进行首次免疫;第一次加强免疫、第二次加强免疫:之后蛋白量减半,按照同样的方法制备重组IFN-γ不完全佛氏佐剂免疫原,每隔14天进行第2次(即第一次加强免疫)、第3次免疫(即第二次加强免疫)。首次免疫采用颈背部皮下多点注射方法;分别在首次免疫和加强免疫前1天以及第二次加强免疫7天后采集尾静脉血,分离血清保存在-20℃待检测。
用包被缓冲液稀释重组IFN-γ蛋白至0.5μg/mL,并包被ELISA板条,待测血清用样本稀释液1/50稀释,100μL/孔,微孔板孵育器37℃孵育45min;弃去一抗,用0.05%PBST,350μL/孔洗涤三次,并在吸水纸上倒扣拍干,加入稀释好的HRP标记二抗,100μL/孔,37℃孵育45min;用0.05% PBST以350μL/孔洗涤3次,并在吸水纸上倒扣拍干;加入ABTS显色液,室温孵育30min;在酶标仪上读数OD405和OD492,OD405-OD492的数值作为ELISA结果。第二次加强免疫后的血清检测结果如表2所示(其中,Y1、Y2、Z1、Z2、ZY表示小鼠编号)。
表2小鼠第2次加强免疫血清测试
实施例4杂交瘤细胞的制备
选择编号为Z1的小鼠为待融合小鼠。在融合前三天,取重组IFN-γ蛋白,50μg/只,腹腔注射Z1小鼠,并观察30min左右。融合当天,取生长状态良好的SP2/0细胞待用,小鼠眼球取血后进行安乐处死,并立即将小鼠置于75%酒精,浸泡5min左右无菌采集脾脏,经研磨后,过200目筛网分离脾细胞,用含10%胎牛血清的1640培养基重悬收集脾细胞并计数,将SP2/0和脾细胞按照一定的比例混匀,离心,收集沉淀,将沉淀团轻柔磕散,在37℃水浴的条件下,依次分别加入PEG1450、1640培养基、含10%胎牛血清的1640培养基,轻柔混匀后,离心收集沉淀,沉淀用一定量的含杂交瘤细胞因子的血清培养基轻柔重悬,按100μL/孔将细胞接种到96孔细胞培养板中,37℃,5% CO2培养箱中培养。
细胞融合步骤完成后,分离Z1小鼠的眼球血,并进行抗IFN-γ血清效价检测。Z1小鼠血清进行间接ELISA法测试,结果如表3所示,抗体滴度可达到1:204800。
表3Z1小鼠抗血清ELISA测试
细胞培养融合后第1天加入4×HAT培养基(含杂交瘤细胞因子和血清),100μL/孔;第3和6天弃掉一半培养基,并加入等量的2×HAT培养基(含杂交瘤细胞因子和血清);待细胞量汇合度达孔底1/3时,可以取孔内一半上清进行间接ELISA法测试,挑出能够识别IFN-γ的阳性杂交瘤细胞。对识别IFN-γ的杂交瘤细胞用有限稀释法稀释,接种至96孔板,使每孔理论上含有1个杂交瘤细胞。连续培养7天左右,选择出孔内只有1个杂交瘤细胞团且OD值高的细胞株,并将此细胞转移到48孔培养板内培养。扩大培养过程中,用间接ELISA法及时监测细胞上清内抗体滴度,最终获得6F7F3D3A11A4C4、6F7D2C4C8H3D3、6F7F3D3A1A6F8这三株杂交瘤细胞株,对杂交瘤细胞6F7F3D3A11A4C4进行保藏,保藏信息如下:杂交瘤细胞株6F7F3D3A11A4C4(Hybridoma cell line6F7F3D3A11A4C4),保藏编号为CCTCC NO:C202358,保藏日期为2023年4月5日。
实施例5腹水法猴IFN-γ单抗的制备及纯化
取6-8周BALB/C雌性小鼠,15只,5只/笼,腹腔注射降植烷,1周后,取实施例4制备得到的三株细胞分别接种小鼠,每组细胞对应1笼小鼠,4只接种,余下1只作为空白对照。注射后7天左右,小鼠腹部膨大,抽取腹水,离心收集上清,每组上清用自制重组IFN-γ蛋白和商业IFN-γ蛋白包被的ELISA板,进行间接ELISA法验证。ELISA测试结果如表4所示,将6F7F3D3A11A4C4腹水记为A组,将6F7D2C4C8H3D3腹水记为B组,将6F7F3D3A1A6F8腹水记为C组;三组腹水的滴度分别为1:102400、1:25600、1:51200。根据ELISA结果,取A组腹水,融化后先后加入乙酸缓冲液和辛酸,混匀后离心,收集上清并透析,最后透析液即为纯化后的抗体,标记为A1。
表4三组腹水效价测试
表5商业试剂测试腹水抗体结果
腹水法猴IFN-γ单抗的SDS-PAGE鉴定:
取出6F7F3D3A11A4C4、6F7D2C4C8H3D3、6F7F3D3A1A6F8这A、B、C三组腹水,均按照2μg/通道的量上样;取A1抗体分别按照1μg/通道,2μg/通道的量上样;加入Loading Buffer,使终浓度为1×溶液,混合后煮沸10min,顺离后取上清20μL上样到10% SurePAGE。用Bio-Rad电泳仪,110V电泳约1h。电泳结束后,将凝胶进行考马斯亮蓝染色,再脱色,直至凝胶透明,透明的凝胶在凝胶成像仪中拍照记录,结果如图2所示。结果显示,A、B、C三组的腹水杂带较多,纯化后的A1抗体,4通道(1μg/通道)的条带,在50kDa处出现目的条带,在25kDa处条带不清晰,5通道(2μg/通道)的条带,在50kDa和25kDa处均出现与预期片段大小相符的条带。
腹水法猴IFN-γ单抗的ELISA鉴定:
对纯化的A1抗体进行间接ELISA法检测,结果显示A1抗体的滴度可达到1:1280,实验结果如表6所示:
表6A1抗体的ELISA鉴定
腹水法猴IFN-γ单抗的Western Blot鉴定:
取一定量的重组IFN-γ蛋白上样,进行SDS-PAGE电泳,电泳结束后,进行湿转膜,转膜结束后,将膜浸没在封闭液中封闭;封闭结束,取出膜备用。取出A1抗体,用稀释液稀释至合适浓度,同时用稀释液做阴性对照,用商业IFN-γ单抗(Rhesus Macaque IFN-gammaBiotinylated Antibody,R&D system,货号P63310)做阳性对照,进行Western Blot。结果如图3所示,A1抗体在20kDa处出现预期(17kDa)的条带(通道2),同时RD标准品的两个浓度也在20kDa处出现预期(17kDa)条带(通道4和通道5),证明本次纯化得到抗体活性良好。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (7)
1.一种分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞株,命名为杂交瘤细胞株6F7F3D3A11A4C4(Hybridoma cellline 6F7F3D3A11A4C4),保藏编号为CCTCCNO:C202358,保藏日期为2023年4月5日,保藏于中国典型培养物保藏中心,保藏地址中国武汉。
2.一种实验猴γ-干扰素单克隆抗体,其特征在于,所述实验猴γ-干扰素单克隆抗体由权利要求1所述的杂交瘤细胞株或其传代细胞株分泌产生。
3.权利要求1所述的杂交瘤细胞株或权利要求2所述的实验猴γ-干扰素单克隆抗体在制备实验猴γ-干扰素的检测试剂方面的应用。
4.一种实验猴γ-干扰素的检测产品,其特征在于,所述实验猴γ-干扰素的检测产品包括权利要求2所述的实验猴γ-干扰素单克隆抗体。
5.如权利要求4所述的检测产品,其特征在于,所述实验猴γ-干扰素的检测产品为试剂、试纸条或试剂盒的至少一种。
6.如权利要求5所述的检测产品,其特征在于,所述试剂盒包括选自下组的至少一种:胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒、微流体芯片试剂盒、荧光免疫试剂盒。
7.如权利要求5所述的检测产品,其特征在于,所述试剂盒包括选自下组的至少一种:双抗体夹心ELISA试剂盒、竞争抑制ELISA检测试剂盒。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310718836.4A CN117024563B (zh) | 2023-06-16 | 2023-06-16 | 分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310718836.4A CN117024563B (zh) | 2023-06-16 | 2023-06-16 | 分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117024563A CN117024563A (zh) | 2023-11-10 |
CN117024563B true CN117024563B (zh) | 2024-04-19 |
Family
ID=88640090
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310718836.4A Active CN117024563B (zh) | 2023-06-16 | 2023-06-16 | 分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117024563B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6683052B1 (en) * | 1998-02-06 | 2004-01-27 | Institut National De La Sante Et De La Recherche Medicale Inserm | Lipopeptides containing an interferon-γ fragment, and uses thereof in pharmaceutical compositions |
CN103361317A (zh) * | 2012-04-06 | 2013-10-23 | 华中农业大学 | 一种猕猴IFN-γ的单克隆抗体杂交瘤细胞及制备方法和应用 |
CN105602908A (zh) * | 2016-01-26 | 2016-05-25 | 中国农业科学院特产研究所 | 水貂γ-干扰素单克隆抗体及其在检测水貂γ-干扰素中的应用 |
CN106434721A (zh) * | 2016-09-26 | 2017-02-22 | 中国人民解放军成都军区疾病预防控制中心 | 用于表达恒河猴干扰素γ的载体和系统 |
CN107083370A (zh) * | 2017-04-14 | 2017-08-22 | 中国医学科学院医学生物学研究所 | 抗树鼩干扰素γ单克隆抗体及分泌该抗体的杂交瘤细胞株与应用 |
-
2023
- 2023-06-16 CN CN202310718836.4A patent/CN117024563B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6683052B1 (en) * | 1998-02-06 | 2004-01-27 | Institut National De La Sante Et De La Recherche Medicale Inserm | Lipopeptides containing an interferon-γ fragment, and uses thereof in pharmaceutical compositions |
CN103361317A (zh) * | 2012-04-06 | 2013-10-23 | 华中农业大学 | 一种猕猴IFN-γ的单克隆抗体杂交瘤细胞及制备方法和应用 |
CN105602908A (zh) * | 2016-01-26 | 2016-05-25 | 中国农业科学院特产研究所 | 水貂γ-干扰素单克隆抗体及其在检测水貂γ-干扰素中的应用 |
CN106434721A (zh) * | 2016-09-26 | 2017-02-22 | 中国人民解放军成都军区疾病预防控制中心 | 用于表达恒河猴干扰素γ的载体和系统 |
CN107083370A (zh) * | 2017-04-14 | 2017-08-22 | 中国医学科学院医学生物学研究所 | 抗树鼩干扰素γ单克隆抗体及分泌该抗体的杂交瘤细胞株与应用 |
Non-Patent Citations (2)
Title |
---|
P63310 • IFNG_MACMU;uniprot;《uniprot》;第1-7页 * |
干扰素γ生物学功能及其应用的研究进展;田园等;《中国生物制品学杂志》(第10期);第118-121页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117024563A (zh) | 2023-11-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110845582B (zh) | 一种猫细小病毒重组蛋白及其单克隆抗体的制备 | |
CN101638660A (zh) | 嗜酸乳杆菌s-层蛋白表面展示系统的构建 | |
CN117024563B (zh) | 分泌实验猴γ-干扰素单克隆抗体的杂交瘤细胞及其应用 | |
CN114276445A (zh) | 轮状病毒重组蛋白特异性抗体、质粒载体及方法 | |
WO2021232800A1 (zh) | 一种泛性型惰性载体大肠杆菌及其潜在应用 | |
CN117384295A (zh) | 小鼠抗鹅IgY单克隆抗体及其应用 | |
CN110257405B (zh) | 牛支原体乙醇脱氢酶基因及其编码蛋白与应用 | |
CN110684097B (zh) | 卡氏住白细胞虫重组r7蛋白及其制备方法和应用 | |
CN1916633A (zh) | 检测梅毒螺旋体的方法及试剂盒 | |
CN104531715A (zh) | 人降钙素原重组表达、单、多克隆抗体的制备及elisa检测方法 | |
CN109932504B (zh) | 用于检测鳜鱼弹状病毒的试剂盒 | |
CN110117325B (zh) | 一种猪cd127多肽及其编码基因和应用 | |
CN111398604A (zh) | 一种牛布病A19-ΔVirB12疫苗的血清学检测方法 | |
CN112730829A (zh) | 一种快速检测牡蛎疱疹病毒OsHV-1的胶体金试纸条及其应用 | |
CN118064376A (zh) | 抗博卡病毒的单克隆抗体及其杂交瘤细胞株和应用 | |
CN114369152B (zh) | 一种重组鸡白细胞介素-9蛋白及其抗体的制备与应用 | |
CN116769019B (zh) | 一种ASFVp30蛋白单克隆抗体及其应用 | |
CN112522210B (zh) | 一种分泌抗小反刍兽疫病毒单克隆抗体的杂交瘤细胞株及其单抗与应用 | |
CN110938127B (zh) | 米氏肉孢子虫抗原、编码基因、重组抗原、试剂盒及应用 | |
CN117384294B (zh) | 小鼠抗鹅IgY单克隆抗体及应用 | |
CN115960184B (zh) | 一种溶血性曼氏杆菌a6血清型白细胞毒素抗原蛋白、其抗体及应用 | |
KR101395932B1 (ko) | 개 브루셀라균 유래 리보솜 단백질 l7/l12 발현 세포주와 발현 단백질을 이용한 진단방법 | |
CN117683142A (zh) | 融合蛋白lsdv090-133及其在检测牛结节性皮肤病病毒抗体中的应用 | |
CN118001377A (zh) | TamA在制备预防和治疗肺炎克雷伯菌感染的产品中的应用 | |
CN118006560A (zh) | 杂交瘤细胞株及其分泌的抗猪cadm1单克隆抗体与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |