CN114940974A - Construction and application of 4-1BB reporter gene 293T stable cell strain - Google Patents
Construction and application of 4-1BB reporter gene 293T stable cell strain Download PDFInfo
- Publication number
- CN114940974A CN114940974A CN202111142531.0A CN202111142531A CN114940974A CN 114940974 A CN114940974 A CN 114940974A CN 202111142531 A CN202111142531 A CN 202111142531A CN 114940974 A CN114940974 A CN 114940974A
- Authority
- CN
- China
- Prior art keywords
- monoclonal
- reporter
- reporter gene
- cell
- stable
- 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.)
- Pending
Links
- 108700008625 Reporter Genes Proteins 0.000 title claims abstract description 53
- 238000010276 construction Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 230000009261 transgenic effect Effects 0.000 claims abstract description 25
- 230000000694 effects Effects 0.000 claims abstract description 22
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 19
- 239000013612 plasmid Substances 0.000 claims abstract description 15
- 238000011156 evaluation Methods 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 7
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 5
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 5
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 28
- 229950010131 puromycin Drugs 0.000 claims description 14
- 210000002966 serum Anatomy 0.000 claims description 14
- 238000011161 development Methods 0.000 claims description 12
- 241000713666 Lentivirus Species 0.000 claims description 11
- 230000027455 binding Effects 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 claims description 7
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 claims description 7
- 229940097277 hygromycin b Drugs 0.000 claims description 7
- 230000012010 growth Effects 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 239000012124 Opti-MEM Substances 0.000 claims description 5
- 239000012096 transfection reagent Substances 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 101150001826 BB gene Proteins 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 32
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 9
- 239000000556 agonist Substances 0.000 abstract description 7
- 230000004913 activation Effects 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 78
- 229940079593 drug Drugs 0.000 description 27
- 108010037274 Member 9 Tumor Necrosis Factor Receptor Superfamily Proteins 0.000 description 18
- 102000011769 Member 9 Tumor Necrosis Factor Receptor Superfamily Human genes 0.000 description 18
- 238000012546 transfer Methods 0.000 description 11
- 108060001084 Luciferase Proteins 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 239000000018 receptor agonist Substances 0.000 description 8
- 229940044601 receptor agonist Drugs 0.000 description 8
- 241000254158 Lampyridae Species 0.000 description 7
- 238000007865 diluting Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 102100040247 Tumor necrosis factor Human genes 0.000 description 4
- 230000005714 functional activity Effects 0.000 description 4
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 3
- 102000002627 4-1BB Ligand Human genes 0.000 description 3
- 108010082808 4-1BB Ligand Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000002380 cytological effect Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000031146 intracellular signal transduction Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 229950005972 urelumab Drugs 0.000 description 2
- 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
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 102000007665 Extracellular Signal-Regulated MAP Kinases Human genes 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 102000004398 TNF receptor-associated factor 1 Human genes 0.000 description 1
- 108090000920 TNF receptor-associated factor 1 Proteins 0.000 description 1
- 102000004393 TNF receptor-associated factor 2 Human genes 0.000 description 1
- 108090000925 TNF receptor-associated factor 2 Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000011990 functional testing Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000003030 reporter gene method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
Images
Classifications
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- 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
- G01N33/6854—Immunoglobulins
-
- 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
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses construction of a 4-1BB reporter gene 293T stable transgenic cell strain, which is characterized in that plasmid containing a 4-1BB gene is used for transfecting monoclonal cells of a stable transgenic cell strain containing an NFkB reporter gene, eukaryotic antibiotics are added for screening and monoclonal selection, and a proper 4-1BB reporter gene stable transgenic cell strain is obtained through functional evaluation. And discloses a method for measuring the biological activity of a 4-1BB agonist medicament by using a 4-1BB reporter gene 293T stable cell strain and a method for measuring a neutralizing antibody, wherein the method can completely represent the 4-1BB activation mechanism and effect or has the advantages of simple detection step, low time cost, low reagent consumable cost and high method stability.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to construction and application of a 4-1BB reporter gene 293T stable transgenic cell strain; and a method for measuring the activity of a CD137 receptor agonist by using the cell line the present invention further relates to a method for detecting a neutralizing antibody of the CD137 receptor agonist.
Background
4-1BB (UniProt ID: Q07011) is a receptor of tumor necrosis factor ligand superfamily member 9(4-1BBL) and belongs to single-transmembrane protein. The 4-1BB receptor and its ligands play a key role in the regulation of the immune response in humans. The 4-1BB receptor is an excitation-induced co-stimulatory molecule, and the 4-1BB receptor activates the function of exerting anti-tumor activity through cytotoxic T cells. The 4-1BBL, after binding to the 4-1BB on the cell membrane, activates the intracellular signaling pathway by recruiting TRAF1 and TRAF2, further activating NF-. kappa.B, AKT, p38MAPK and ERK signaling pathways.
4-1 BB-targeted agonist modulators are the hot spots of current development in the development of tumor immunotherapy drugs. No drug targeting 4-1BB is currently on the market, and 4-1BB agonists in clinical development stage include more than ten products such as Urelumab (BMS-663513), Utomillumab (PF-05082566), LVGN-6051, ADG-106 and ES101, wherein monoclonal antibodies and bispecific antibodies exist. The medicine targeted to 4-1BB has wide research and development prospect and strong competition, and the biological analysis method (including an activity test method and a neutralizing antibody detection method) of the 4-1BB agonist is an important tool for the research and development of the targeted 4-1BB medicine and has a great market application scene.
The prior art has the following defects: the development of biological therapeutic drugs mainly comprises three major stages of drug discovery, preclinical and clinical research and commercial production. The determination of the biological activity of a drug relative to a target is an essential process in drug discovery and commercial production. 4-1BB receptor protein can be expressed by recombination under normal conditions, and the biological activity of the drug can be judged by ELISA technology through the binding activity test of the 4-1BB candidate drug and the 4-1BB receptor protein. Naturally, 4-1BB exists on the surface of a cell membrane in a homotrimeric state, binding of the candidate drug and 4-1BB at the protein level does not necessarily activate 4-1BB receptors to initiate downstream signaling pathways, and it is described in the literature that the candidate drug activates intracellular signaling pathways to exert physiological functions only when the candidate drug binds to and multimerizes at the surface of the cell membrane 4-1BB receptors. Under a real physiological environment, 4-1BB on the surfaces of cytotoxic T cells can be activated by candidate drugs so as to play a role in killing the T cells, and if the functional activity of the cytotoxic T cells is tested by collecting whole blood or separating PBMC (peripheral blood mononuclear cell) and incubating the PBMC with the candidate drugs, the proliferation detection of the T cells or the cytokine release of an incubation supernatant is tested by using a flow technology. The activity of the 4-1BB candidate drug is determined by an ELISA method based on the ligand-receptor binding principle, and the functional activity of the candidate drug and the target 4-1BB receptor is not characterized only by determining the binding activity of the candidate drug and the target. The whole blood or PBMC is used for functional activity characterization, the detection process is complicated, the individual difference of the whole blood or PBMC is large, the batch difference is large, and the stability of the method is poor. The 4-1BB reporter gene 293T stable transgenic cell strain is taken as a key reagent, and the activity test of the candidate drug at a cell level can characterize the functional activity of the candidate drug, and meanwhile, the stability of the method is very excellent, so that the method is a relatively suitable activity determination method of the 4-1BB candidate drug.
The detection of anti-drug antibodies and neutralizing antibodies is an essential step in the assessment of drug safety and efficacy in preclinical and clinical studies. The platforms of neutralizing antibody (NAb) assay methods can be largely divided into cell-based and non-cell based. Because of the better correlation between cytological methods and the physiology of the in vivo environment of the drug, the EMA, FDA and CFDA immunogenicity assay guidelines all show their preference for cytological neutralizing antibody detection. The 4-1BB reporter gene 293T stable transfer cell strain is taken as a key reagent, and the detection of the neutralizing antibody of the candidate drug at the cell level has physiological relevance compared with the detection of the neutralizing antibody at the protein level of a ligand receptor binding principle, and meets the requirements of regulatory supervision and guidance principles.
Disclosure of Invention
The invention aims to provide construction and application of a 4-1BB reporter gene 293T stable transgenic cell strain, which solve the problems that a biological activity determination method and a neutralizing antibody determination method in the development process of a 4-1BB agonist drug cannot completely characterize a 4-1BB activation mechanism and effect or detection steps are complicated, time and cost are high, reagent consumable cost is high, method stability is poor and the like.
According to the construction of the 4-1BB reporter gene 293T stable transgenic cell strain provided by the invention, a plasmid containing the 4-1BB gene is used for transfecting a monoclonal cell of the stable transgenic cell strain containing the NFkB reporter gene, and a proper 4-1BB reporter gene stable transgenic cell strain is obtained through adding eukaryotic antibiotics for screening and monoclonal selection and functional evaluation.
In some embodiments, the specific construction steps are as follows: add 20ug 4-1BB gene plasmid to 388.3 μ L of Opti-MEM; mixing well, then adding 20 mu L X-tremagene transfection reagent, mixing well, and standing for half an hour at room temperature. The plasmid mixed solution is added into a 10cm plate containing 5ml of DMEM with about 50% confluency of the NFKB reporter gene monoclonal cell strain functional monoclonal KB-20, the mixture is uniformly mixed, the cell is changed on the third day, and 1 mu g/ml puromycin and 200 mu g/ml Hygromycin B are added into DMEM culture medium for pressure culture. After the growth and passage of 1 mug/ml puromycin and 200 mug/ml Hygromycin B for 2 times, the cells are paved with 1/hole, 200 ul/hole is used for 96-hole plate monoclonal paving, the cell condition of each hole is observed under a microscope after the 96-hole plate monoclonal paving is carried out for 14 days, the selected monoclonal enters a 24-hole plate, the 24-hole plate cells are transferred to a 6-hole plate after 1-week culture, and then the cells are subjected to T75 and T175 amplification culture to obtain the better monoclonal NC 86.
In some embodiments, the 4-1BB gene plasmid is sinobiological, cat. hg100414-1 BB gene SEQ ID No. 2; the Opti-MEM is in a model of Thermo Fisher, Cat.31985-070; the model of the X-tremeGENE transfection reagent is Roche, Cat.6366244001.
In some embodiments, 293T cells are transfected with lentiviruses containing NFkB reporter genes, screened by adding eukaryotic antibiotics and selected for monoclonals, and selected to obtain suitable stable NFkB reporter gene transfer cell strains monoclonals after functional evaluation
In some embodiments, the NFKB reporter monoclonal cell strain is constructed by infecting 293T cells at 2E 5/well in 6-well plates with the NFKB reporter lentivirus group a MOI of 5 and group B MOI of 1 in a total volume of 1000 ul; cell liquid change is carried out the next day, and the lentivirus is removed; on the fourth day, the digested cells were transferred to a T75 cell culture flask and added to 1640 medium of 1ug/ml puromycin for pressure culture; after maintaining 1ug/ml puromycin 1640 medium for growth, passaging and pressurizing for two generations, the cells were plated at 1/well in 96-well plates on day 8; on day 19, the single clones observed in the 96-well plate were transferred to a 24-well plate, and after evaluation screening, transferred to a 6-well plate, T25, T75, and T175, and cultured to obtain single clones.
In some embodiments, the NFkB reporter lentivirus is of the type kindly customized, and the DNA sequence of the NFkB binding site and miniromoter in the NFkB reporter is set forth in SEQ ID No. 1.
In some embodiments, the 293T cell is of the type ATCC, cat # CRL-3216.
The application of the construction of 4-1BB reporter gene 293T stable transgenic cell strain in the development and determination of 4-1BB activity determination method.
An application of a 4-1BB reporter gene 293T stable transfer cell strain in human serum tolerance determination.
An application of 4-1BB reporter gene 293T stable transgenic cell strain in the development of 4-1BB neutralizing antibody detection method.
Has the advantages that:
reporter genes such as the luciferase gene (luciferase) are often used to characterize intracellular transcriptional activity by detecting the activity of proteins or enzymes translated from the reporter gene. The effect of a promoter or enhancer on gene expression can be measured by specific experimental tests of the reporter gene. In the firefly enzyme detection method, firefly enzyme takes ATP, oxygen and firefly enzyme luciferin as substrates, a chemical reaction is catalyzed, light can be detected by an instrument when released, and the intensity of chemiluminescence can represent the quantity of the firefly enzyme. Compared with other fluorescent reporter gene methods, the firefly enzyme reporter gene detection method has extremely low background signal and extremely high sensitivity, the lower limit of the concentration of the detected firefly enzyme can reach the number level of 10-11M, and the firefly enzyme reporter gene detection method is widely applied to the research of intracellular signal paths. The invention constructs a 4-1BB reporter gene stable transfer cell strain, and additionally inserts a luciferase gene expression system to form the 4-1BB reporter gene stable transfer cell strain, wherein the luciferase gene expression system comprises three parts, namely an NFkB transcription factor nucleotide binding site, a miniprometer and a luciferase gene, the activation effect of 4-1BB excitation can be characterized by the activity of a transcription factor NFkB at the downstream of a signal path, and the luciferase gene expression system can be combined with the active transcription factor NFkB to start the expression of luciferase, so that the activation effect of 4-1BB agonist can be characterized by detecting the amount of luciferase, and the method is further used for measuring the activity of 4-1BB receptor agonist, and a quantitative method for the 4-1BB receptor agonist by using the cell strain. Further relates to a method for measuring the neutralizing antibody of the 4-1BB receptor agonist. Further relates to a method for measuring the neutralizing antibody of the 4-1BB receptor agonist. In view of the high sensitivity of the luciferase detection method, the method has extremely high sensitivity, the 4-1BB reporter gene stable transfer cell strain is characterized by the activity of a transcription factor NFkB of a downstream signal molecule of a 4-1BB signal channel, the signal transmission is deeper, the specificity is further enhanced, and the anti-interference capability is enhanced. The natural 4-1BB receptor on the surface of the cell membrane exists in a homotrimerization mode, the 4-1BB receptor on the surface of the cell membrane needs to be activated, the candidate drug can be combined only under the precondition, the candidate drug also needs to enable the 4-1BB receptor to be gathered on the surface of the cell membrane to activate the signal path of the cell, and the functional test of the stable 4-1BB reporter gene transfer cell strain is suitable for being closer to the action mechanism of the 4-1BB receptor agonist drug.
The method for analyzing the drug concentration by adopting the 4-1BB reporter gene stable transfer cell strain is a universal method, can be used for performing activity test on all 4-1BB agonists/candidate drugs, and has good stability and high sensitivity. The method for detecting the neutralizing antibody is a part of medicine safety and effectiveness, the neutralizing antibody detection method is obtained by adjusting and converting conditions of an activity detection method in a medicine discovery process, a detection system is basically consistent with the activity detection method, and the method is a better choice for constructing a 4-1BB reporter gene stable transfer cell strain and detecting the neutralizing antibody of a 4-1BB receptor agonist by using a fluorescence report detection system.
Drawings
FIG. 1 shows the results of the test of the transfection efficiency evaluation of NFKB reporter plasmid;
FIG. 2 shows the results of the functional evaluation of the NFkB reporter monoclonal cell strain;
FIG. 3 shows the results of the development and measurement of 4-1BB activity of 4-1BB reporter gene-transfected cell line;
FIG. 4 is the results of a dose curve experiment for the determination of human serum tolerance of 4-1BB reporter gene stable transgenic cell line;
FIG. 5 is a distribution of ratios of individual sera of 4-1BB reporter transfected cell lines used in 4-1BB neutralizing antibody detection method.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Construction process of NFKB reporter gene stable transgenic cell strain
NFkB reporter lentivirus (Kingson custom, NFkB binding site in NFkB reporter and DNA sequence of miniromoter see SEQ ID NO: 1) group A MOI of 5 and group B MOI of 1 293T cells (ATCC, cat # CRL-3216) infected at 2E 5/well in 6-well plates in a total volume of 1000 ul; cell change is carried out the next day, and the lentivirus is removed; on the fourth day, the digested cells were transferred to a T75 cell culture flask and added to 1640 medium at 1ug/ml puromycin for pressure culture. After maintaining 1ug/ml puromycin 1640 medium for growth, passaging and pressurizing for two generations, the cells were plated at 1/well in 96-well plates on day 8; on day 19, the single clones observed in the 96-well plate were transferred to a 24-well plate, and after evaluation screening, transferred to a 6-well plate, T25, T75, and T175, and cultured to obtain single clones.
1 NFkB binding site and minimaster DNA sequence
GGGAATTTCCGGGGACTTTCCGGGAATTTCCGGGGACTTTCCGGGAATTTCCAGATCTGGCCTCGGCGGCCAAGCTTAGACACTAGAGGGTATATAATGGAAGCTCGACTTCCAG
Example 2
Evaluation of transfection efficiency of NFKB reporter plasmid
293T cells transfected by reporter lentivirus are taken, pressurized and cultured for two generations by 1 mu g/mL puromycin DMEM, 80uL of digested cell suspension (10000 cells/hole) is taken to each hole of a white impervious 96-well plate, 20uL of PMA with the concentration of 500nM and 20uL of TNF-alpha with the concentration of 500ng/mL or 20uL of culture medium are added, and after uniform mixing, the mixture is placed into an incubator with 37 ℃ and 5% CO2 for culture and incubation for 5 hours. Adding 100ul Bright-Glo TM Luciferase Assay reagent (Promega E2620) was mixed uniformly at 8000rpm for 5 minutes at room temperature and placed in a chemiluminescence detector for detection.
The detection results are shown in FIG. 1, after the signal value of TNF alpha treatment is relative to the background group of the culture medium treatment, the transfected cells have strong signal response and signal to noise ratio which is close to 10 times, and because the receptor of TNFalpha naturally exists on the surface of 293T cells, the main intracellular signal path after the receptor of TNFalpha is activated into the signal path containing NFkB, and the results show that the NFkB reporter gene is inserted into the 293T cells and can be effectively activated.
Example 3
Function evaluation of NFkB reporter gene monoclonal cell strain in 24-well plate
Selecting monoclonal from a 96-well plate paved with the monoclonal, transferring the monoclonal to a 24-well plate, after the clone in the 24-well plate is basically overgrown, after 100ul of trypsinized cells are digested for 2-3 minutes, adding 400ul of culture medium for resuspending the cells, taking 80 mu L/well to a well of a white impervious microplate, dividing the well into 2 wells, respectively adding 20 mu L of TNF-alpha with the concentration of 500ng/mL or 20ul of culture medium into the well, uniformly mixing, and putting the well into an incubator with 37 ℃ and 5% CO2 for culture and incubation for 5 hours.
Adding 100ul Bright-Glo TM And (3) uniformly mixing the Luciferase Assay reagent at room temperature of 1000rpm for 5 minutes, and placing the mixture into a chemiluminescence detector for detection. The detection results of the monoclonals are shown in figure 2, the experimental results show that different monoclonals are treated by culture medium or TNF-alpha, the generated signal values have larger difference, clone KB-20 with better TNFalpha/culture medium signal noise is selected for further amplification culture, and the clone KB-20 is used as a mother cell for constructing 4-1BB reporter gene stable cells
Example 4
4-1BB reporter gene stable transfer cell strain construction process
20ug 4-1BB gene plasmid (sinobiological, Cat. HG100414-1 BB gene SEQ ID NO: 2) was mixed with 388.3 μ L Opti-MEM (Thermo Fisher, Cat.31985-070) and 20 μ L X-tremagene transfection reagent (Roche, Cat.6366244001) was added and mixed well for half an hour at room temperature. The plasmid mixture was added to a 10cm dish containing 5ml of DMEM with about 50% confluency of the functional monoclonal KB-20 of the NFKB reporter monoclonal cell strain, cell exchange was performed on the third day and pressure culture was performed by adding 1. mu.g/ml puromycin and 200. mu.g/ml Hygromycin B in DMEM medium. After the growth and passage of 1 mug/ml puromycin and 200 mug/ml Hygromycin B for 2 times, the cells are paved with 1/hole, 200 ul/hole is used for 96-hole plate monoclonal paving, the cell condition of each hole is observed under a microscope after the 96-hole plate monoclonal paving is carried out for 14 days, the selected monoclonal enters a 24-hole plate, the 24-hole plate cells are transferred to a 6-hole plate after 1-week culture, and then the cells are subjected to T75 and T175 amplification culture to obtain the better monoclonal NC 86.
24-1 BB DNA sequence of SEQ ID no
atgggaaacagctgttacaacatagtagccactctgttgctggtcctcaactttgagaggacaagatcattgcaggatccttgtagtaactgcccagctggtacattctgtgataataacaggaatcagatttgcagtccctgtcctccaaatagtttctccagcgcaggtggacaaaggacctgtgacatatgcaggcagtgtaaaggtgttttcaggaccaggaaggagtgttcctccaccagcaatgcagagtgtgactgcactccagggtttcactgcctgggggcaggatgcagcatgtgtgaacaggattgtaaacaaggtcaagaactgacaaaaaaaggttgtaaagactgttgctttgggacatttaacgatcagaaacgtggcatctgtcgaccctggacaaactgttctttggatggaaagtctgtgcttgtgaatgggacgaaggagagggacgtggtctgtggaccatctccagccgacctctctccgggagcatcctctgtgaccccgcctgcccctgcgagagagccaggacactctccgcagatcatctccttctttcttgcgctgacgtcgactgcgttgctcttcctgctgttcttcctcacgctccgtttctctgttgttaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgtga
Example 5
4-1BB transfection efficiency assessment
Cells after 4-1BB gene plasmid transfection and pressure culture by 1 mug/ml puromycin and 200 mug/ml Hygromycin B are taken, 100ul pancreatin digestion is carried out in a 24-well plate, 400ul culture medium is resuspended, 100ul cell suspension is taken, 5ul Anti-CD137 Antibody is added to the cell suspension to achieve a final concentration of 5ug/ml, bubble generation is reduced as much as possible in the mixing process, the cell suspension is incubated at 4 ℃ for at least 30min, a proper amount of PBS is taken out and added for centrifugation at 1000rpm for 5min, supernatant is discarded, the operation is repeated for 2 times, 100ul samples are remained, 5ul Anti-Fc fluorescent Antibody is added, the cell suspension is incubated at the final concentration of 5ug/ml and 4 ℃ for 30min, a proper amount of PBS is taken out and added for centrifugation at 1000rpm for 5min, supernatant is discarded, the operation is repeated for 2 times, 20ul is taken and added into a cell counting plate, and RFP is counted.
According to the result of the cell counter, the positive rate is 73%, which shows that most of the 4-1BB receptor protein in the cell population is expressed with high kurtosis after the 4-1BB gene plasmid is transfected and is cultured under pressure.
Example 6
Development and determination of 4-1BB activity assay method of 4-1BB reporter gene stable transgenic cell strain
Selecting a 4-1BB reporter gene stable transgenic cell strain, namely, a monoclonal NC86 strain, a monoclonal NC 4 strain per well, and a monoclonal NC 96-well strain, wherein 80 ul/well are planted in a white 96-well plate which is impermeable to the bottom; diluting 4-1BB anti-4-1 BB antibody (customized by Baiying Biotech Co., Ltd., Tanzhou, cat # B6241) from an initial working concentration of 2000. mu.g/ml, diluting 10 spots by 2-fold, adding 20ul into a well containing a stable transgenic cell strain clone NC86, mixing well, incubating in an incubator at 37 ℃ and 5% CO25 hours, 50ul of Bright-Lumi was added TM And (3) uniformly mixing the Luciferase Assay reagent at room temperature of 800rpm for 5 minutes, and placing the mixture into a chemiluminescence detector for detection.
Detailed results As shown in FIG. 3, the signal value generated by the 4-1BB reporter gene stable transgenic cell line is gradually increased along with the increase of the concentration of the 4-1BB antibody, and a remarkable dose response curve is obtained, so that the method can be used for measuring the activity of the 4-1BB antibody.
Example 7
4-1BB reporter gene stable transfer cell strain human serum tolerance determination
Selecting a 4-1BB reporter gene stable transgenic cell strain, namely, a monoclonal NC86 strain, a monoclonal NC 4 strain per well, and a monoclonal NC 96-well strain, wherein 80 ul/well are planted in a white 96-well plate which is impermeable to the bottom; diluting human serum with serum-free DMEM medium to obtain dilutions with human serum content of 20%, 10% and 5%, respectively, diluting anti-4-1 BB antibody with the above three dilutions, performing gradient dilution from initial working concentration of 1000 μ g/ml, diluting 10 points by 2 times, adding 20ul into the well containing stable transgenic cell strain clone NC86, mixing well, culturing in 37 deg.C 5% CO2 incubator for 5 hr, adding 50ul Bright-Lumi TM And (3) uniformly mixing the Luciferase Assay reagent at room temperature of 800rpm for 5 minutes, and placing the mixture into a chemiluminescence detector for detection. The results of the dose profile experiment are shown in figure 4. In the detection system, the reaction matrix is culture medium or 5% of human Pooled serum, and a similar dose response curve is provided, which indicates that the monoclonal NC86 of the 4-1BB reporter gene stable cell line is tolerant to 5% of human serum and can be used for sample analysis and detection in human serum matrix environment.
Example 8
4-1BB reporter gene stable transgenic cell strain for development of 4-1BB neutralizing antibody detection method
Selecting a 4-1BB reporter gene stable transgenic cell strain, namely, a monoclonal NC86 strain, a monoclonal NC 4 strain per well, and a monoclonal NC 96-well strain, wherein 80 ul/well are planted in a white 96-well plate which is impermeable to the bottom; diluting the anti-4-1 BB antibody to initial working concentration of 3200ng/ml with a culture medium, adding into human individual serum or human mixed serum, diluting in half, and standing at room temperature for 15 min; adding the incubated solution into 80 ul/well cell suspension at 20 ul/well, mixing, culturing in a 37 deg.C incubator containing 5% CO2 for 5 hr, adding 50ul Bright-Lumi TM Luciferase AsAnd (5) uniformly mixing the say reagent at room temperature of 800rpm for 5 minutes, and placing the mixture into a chemiluminescence detector for detection. The ratio distribution of the individual serum is shown in figure 5 by carrying out statistical analysis on the ratio of the human individual serum/human mixed serum (NC), and the cut point of the 4-1BB neutralizing antibody detection method is preliminarily determined to be 0.887.
The foregoing is only a preferred form of the invention and it should be noted that numerous similar variations and modifications could be made by those skilled in the art without departing from the inventive concept herein, which shall be considered to be within the scope of the appended claims.
Claims (9)
1. A4-1 BB reporter gene 293T stable transgenic cell strain construction is characterized in that a plasmid containing a 4-1BB gene is used for transfecting a monoclonal cell of a stable transgenic cell strain containing an NFkB reporter gene, and a proper 4-1BB reporter gene stable transgenic cell strain is obtained through adding eukaryotic antibiotics for screening and monoclonal selection and functional evaluation.
2. The construction of the stable 4-1BB reporter gene 293T cell line of claim 1, which comprises the following specific construction steps: add 20ug 4-1BB gene plasmid to 388.3 μ L of Opti-MEM; mixing well, then adding 20 mu L X-tremeGENE transfection reagent, mixing well and standing for half an hour at room temperature. The plasmid mixture was added to a 10cm dish containing 5ml of DMEM with about 50% confluency of the functional monoclonal KB-20 of the NFKB reporter monoclonal cell strain, cell exchange was performed on the third day and pressure culture was performed by adding 1. mu.g/ml puromycin and 200. mu.g/ml Hygromycin B in DMEM medium. After the growth and passage of 1 mug/ml puromycin and 200 mug/ml Hygromycin B for 2 times, the cells are paved with 1/hole, 200 ul/hole is used for 96-hole plate monoclonal paving, the cell condition of each hole is observed under a microscope after the 96-hole plate monoclonal paving is carried out for 14 days, the selected monoclonal enters a 24-hole plate, the 24-hole plate cells are transferred to a 6-hole plate after 1-week culture, and then the cells are subjected to T75 and T175 amplification culture to obtain the better monoclonal NC 86.
3. The construction of the 4-1BB reporter 293T stably transfected cell line of claim 2, wherein the 4-1BB gene plasmid is sinoviral, Cat.HG100414-1 BB gene SEQ ID NO. 2; the Opti-MEM is in a model of Thermo Fisher, Cat.31985-070; the model of the X-tremeGENE transfection reagent is Roche, Cat.6366244001.
4. The construction of the stable 4-1BB reporter gene 293T cell line according to claim 1, wherein a lentivirus containing NFkB reporter gene is used for transfecting 293T cells, eukaryotic antibiotics are added for screening and monoclonal selection, and through functional evaluation, a proper single clone of the stable NFkB reporter gene cell line is selected.
5. The construction of the stable 4-1BB reporter 293T cell line of claim 4, wherein the NFKB reporter monoclonal cell line is constructed by infecting 293T cells at 2E 5/well in a 6-well plate with NFkB reporter lentivirus group A MOI of 5 and group B MOI of 1 in a total volume of 1000 ul; cell liquid change is carried out the next day, and the lentivirus is removed; on the fourth day, the digested cells were transferred to a T75 cell culture flask and added to 1640 medium of 1ug/ml puromycin for pressure culture; after two passages of pressure for growth and subculture in 1ug/ml puromycin 1640 medium, cells were plated in 96-well plates at day 8; on day 19, the single clones observed in the 96-well plate were transferred to a 24-well plate, and after evaluation screening, transferred to a 6-well plate, T25, T75, and T175, and cultured to obtain single clones.
6. The construction of the 4-1BB reporter 293T stable cell line of claim 5, wherein the model of the NFkB reporter lentivirus is Kingchi custom, and the DNA sequence of the NFkB binding site and minimaster in the NFkB reporter is shown in SEQ ID No. 1.
7. The use of the construction of a stable cell line of 293T reporter gene 4-1BB as claimed in any of claims 1-6 in the development and determination of a method for determining the activity of 4-1 BB.
8. Use of a 4-1BB reporter 293T stably transfected cell line of any of claims 1-6 in human serum tolerance assay.
9. The use of the 4-1BB reporter 293T stably transfected cell line of any of claims 1-6 in the development of a 4-1BB neutralizing antibody detection method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111142531.0A CN114940974A (en) | 2021-09-28 | 2021-09-28 | Construction and application of 4-1BB reporter gene 293T stable cell strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111142531.0A CN114940974A (en) | 2021-09-28 | 2021-09-28 | Construction and application of 4-1BB reporter gene 293T stable cell strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114940974A true CN114940974A (en) | 2022-08-26 |
Family
ID=82905928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111142531.0A Pending CN114940974A (en) | 2021-09-28 | 2021-09-28 | Construction and application of 4-1BB reporter gene 293T stable cell strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114940974A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492657A (en) * | 2011-12-16 | 2012-06-13 | 兰州大学 | Drug screening cell model using NF-kappa B as target and building and applications thereof |
| CN105925544A (en) * | 2016-06-27 | 2016-09-07 | 武汉思安医疗技术有限公司 | Preparation method and application of 4-1BB-containing lentivirus |
| CN108823640A (en) * | 2018-06-06 | 2018-11-16 | 珠海铂华生物工程有限公司 | A kind of method and its application constructing the high-throughput sequencing library based on lymthoma genetic test |
| CN109837249A (en) * | 2017-11-28 | 2019-06-04 | 南京传奇生物科技有限公司 | For evaluating the active recombinant cell of anti-tnf-alpha pharmaceutical biology and its application |
| CN110499295A (en) * | 2019-09-11 | 2019-11-26 | 宝船生物医药科技(上海)有限公司 | A kind of CSF-1R reporter gene cell strain and its preparation method and application |
| US20200325225A1 (en) * | 2016-12-19 | 2020-10-15 | Hoffmann-La Roche Inc. | Combination therapy with targeted 4-1bb (cd137) agonists |
| US20210130845A1 (en) * | 2017-09-08 | 2021-05-06 | Poseida Therapeutics, Inc. | Compositions and methods for chimeric ligand receptor (clr)-mediated conditional gene expression |
-
2021
- 2021-09-28 CN CN202111142531.0A patent/CN114940974A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492657A (en) * | 2011-12-16 | 2012-06-13 | 兰州大学 | Drug screening cell model using NF-kappa B as target and building and applications thereof |
| CN105925544A (en) * | 2016-06-27 | 2016-09-07 | 武汉思安医疗技术有限公司 | Preparation method and application of 4-1BB-containing lentivirus |
| US20200325225A1 (en) * | 2016-12-19 | 2020-10-15 | Hoffmann-La Roche Inc. | Combination therapy with targeted 4-1bb (cd137) agonists |
| US20210130845A1 (en) * | 2017-09-08 | 2021-05-06 | Poseida Therapeutics, Inc. | Compositions and methods for chimeric ligand receptor (clr)-mediated conditional gene expression |
| CN109837249A (en) * | 2017-11-28 | 2019-06-04 | 南京传奇生物科技有限公司 | For evaluating the active recombinant cell of anti-tnf-alpha pharmaceutical biology and its application |
| CN108823640A (en) * | 2018-06-06 | 2018-11-16 | 珠海铂华生物工程有限公司 | A kind of method and its application constructing the high-throughput sequencing library based on lymthoma genetic test |
| CN110499295A (en) * | 2019-09-11 | 2019-11-26 | 宝船生物医药科技(上海)有限公司 | A kind of CSF-1R reporter gene cell strain and its preparation method and application |
Non-Patent Citations (8)
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deniger et al. | Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor | |
| CN110691844B (en) | Methods and kits for generating simulated innate immune cells from pluripotent stem cells | |
| Yen et al. | Facile discovery of surrogate cytokine agonists | |
| CN112538461B (en) | GLP1R reporter gene stable transgenic cell strain, construction method and application | |
| JP2022541181A (en) | Method for isolating TCR gene | |
| CN114480504A (en) | Construction and application of Claudin18.2 reporter gene CHO-K1 stable transgenic cell strain | |
| Lanigan et al. | Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system | |
| Straub et al. | Recruitment of epitope-specific T cell clones with a low-avidity threshold supports efficacy against mutational escape upon re-infection | |
| Lisby et al. | Evaluation of CAR-T cell cytotoxicity: Real-time impedance-based analysis | |
| US20080132427A1 (en) | Cell systems and methods for detecting proliferation acitvity | |
| US20220081691A1 (en) | Modulating ptpn2 to increase immune responses and perturbing gene expression in hematopoietic stem cell lineages | |
| CN107384869A (en) | A kind of monoclonal cell strain and its application in IL 6R inhibitor relative biological activities are determined | |
| RU2139936C1 (en) | Method of determining modulation effect of substance on interleukin-5 receptor-linked signal-transmission route in human or animal cell | |
| CN114940974A (en) | Construction and application of 4-1BB reporter gene 293T stable cell strain | |
| CN109837249A (en) | For evaluating the active recombinant cell of anti-tnf-alpha pharmaceutical biology and its application | |
| CN113337543A (en) | GCGR reporter gene stable-transfer cell strain and construction method and application thereof | |
| CN114231493A (en) | Construction and application of GIPR reporter gene stable cell strain | |
| CN112921004B (en) | Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method | |
| Yang et al. | Proviral integration site 2 is required for interleukin‐6 expression induced by interleukin‐1, tumour necrosis factor‐α and lipopolysaccharide | |
| Reuven et al. | Functional Assays Evaluating Immunosuppression Mediated by Myeloid‐Derived Suppressor Cells | |
| Prawiro et al. | A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells | |
| CN108570450A (en) | A kind of application of cell model and its construction and screening Serotonin receptor target active substance | |
| CN102888382A (en) | Cell model, construction method and application thereof for screening nervous disorder resistant medicines | |
| CN103917645A (en) | Cell free translation system for compound screening and related uses | |
| CN111500668A (en) | Method for determining biological activity of human I L-36/I L36R/I L1 RAcP pathway inhibitor |
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 |