CN115044612A - African swine fever virus p72 protein stable expression cell line and construction method thereof - Google Patents
African swine fever virus p72 protein stable expression cell line and construction method thereof Download PDFInfo
- Publication number
- CN115044612A CN115044612A CN202210699860.3A CN202210699860A CN115044612A CN 115044612 A CN115044612 A CN 115044612A CN 202210699860 A CN202210699860 A CN 202210699860A CN 115044612 A CN115044612 A CN 115044612A
- Authority
- CN
- China
- Prior art keywords
- protein
- expression
- cell line
- swine fever
- african swine
- 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
- 101710121996 Hexon protein p72 Proteins 0.000 title claims abstract description 79
- 101710125418 Major capsid protein Proteins 0.000 title claims abstract description 79
- 241000701386 African swine fever virus Species 0.000 title claims abstract description 45
- 230000010473 stable expression Effects 0.000 title claims abstract description 21
- 238000010276 construction Methods 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 55
- 230000014509 gene expression Effects 0.000 claims abstract description 42
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 37
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 37
- 239000013604 expression vector Substances 0.000 claims abstract description 27
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 25
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims abstract description 5
- 239000002244 precipitate Substances 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 11
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 11
- 229930027917 kanamycin Natural products 0.000 claims description 10
- 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 claims description 10
- 229960000318 kanamycin Drugs 0.000 claims description 10
- 229930182823 kanamycin A Natural products 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 238000001262 western blot Methods 0.000 claims description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 239000012880 LB liquid culture medium Substances 0.000 claims description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 8
- 239000001569 carbon dioxide Substances 0.000 claims description 8
- 230000006037 cell lysis Effects 0.000 claims description 8
- 235000014304 histidine Nutrition 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 238000010367 cloning Methods 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 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 claims description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 102000037865 fusion proteins Human genes 0.000 claims description 6
- 108020001507 fusion proteins Proteins 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 5
- 241000282898 Sus scrofa Species 0.000 claims description 5
- 238000005336 cracking Methods 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- 239000002502 liposome Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000012521 purified sample Substances 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 208000007407 African swine fever Diseases 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 3
- 108091081024 Start codon Proteins 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000013592 cell lysate Substances 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000012091 fetal bovine serum Substances 0.000 claims description 3
- 150000002411 histidines Chemical class 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 210000004748 cultured cell Anatomy 0.000 claims description 2
- 238000003119 immunoblot Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 230000007480 spreading Effects 0.000 claims description 2
- 238000003892 spreading Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims 2
- 239000000523 sample Substances 0.000 claims 2
- 108010033276 Peptide Fragments Proteins 0.000 claims 1
- 102000007079 Peptide Fragments Human genes 0.000 claims 1
- 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 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 229950010131 puromycin Drugs 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 101800000958 Reverse transcriptase/ribonuclease H Proteins 0.000 description 6
- 241000282887 Suidae Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000012795 verification Methods 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
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- 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
- C12N2510/02—Cells for production
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/12011—Asfarviridae
- C12N2710/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/12011—Asfarviridae
- C12N2710/12051—Methods of production or purification of viral material
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
- C12N2840/105—Vectors comprising a special translation-regulating system regulates levels of translation enhancing translation
-
- 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
-
- 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
- C12R2001/91—Cell lines ; Processes using cell lines
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a stable expression cell line of African swine fever virus p72 protein and a construction method thereof, wherein after the sequence transformation of the p72 protein is completed to obtain recombinant protein, the recombinant protein is respectively cloned to a prokaryotic expression vector and an eukaryotic expression vector to respectively carry out prokaryotic expression and eukaryotic expression; wherein, in the process of prokaryotic expression, escherichia coli which stably expresses p72 recombinant protein and purified p72 recombinant protein can be obtained; after eukaryotic expression, a CHO cell strain capable of stably expressing p72 recombinant protein can be obtained. The construction method can be used for screening and obtaining the CHO cell strain expressing the African swine fever virus p72 protein within 2 months, and the cell line is transmitted to 20 generations without puromycin screening pressure, so that the exogenous recombinant protein can be still detected, the success rate is 90%, and the construction method has the advantages of short test period, high success rate and low expression cost.
Description
Technical Field
The invention relates to the technical field of recombinant protein expression, in particular to a stable expression cell line of African swine fever virus p72 protein and a construction method thereof.
Background
African Swine fever (African Swine fever virus) is an acute, hemorrhagic, or virulent infectious disease caused by African Swine fever virus (African Swine fever virus) infecting domestic pigs and various wild pigs (such as African wild pigs, European wild pigs, etc.). The world animal health organization classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a kind of animal epidemic situation which is mainly prevented in China. It is characterized by short disease course and the death rate of the most acute and acute infections up to 100%.
The African swine fever virus shell is an icosahedral capsid and is mainly assembled by a protein P72 encoded by a virus gene. The Major Capsid Protein (MCP) P72 is the most important structural component of the virus, accounting for about 31% -33% of the total mass of the virus, making it one of the major antigens of infected pigs. Research data show that the monoclonal antibody recognizing P72 can neutralize ASFV isolate with strong toxicity, the P72 protein is relatively conservative and has stable antigenicity, and the antibody aiming at P72 can inhibit the combination of virus and macrophage in connection with the process of virus entering host cell. Thus, p72 is a very good protective antigen.
Prokaryotic protein expression systems are both the most commonly used and the most cost-effective protein expression systems. The prokaryotic protein expression system is represented by an escherichia coli expression system, and has the advantages of clear genetic background, low cost, high expression quantity, relatively simple separation and purification of an expression product and the like. The major advantage of mammalian cell expression systems is that protein post-translational processing mechanisms are closest to the native form in vivo, most readily retaining biological activity, and Chinese Hamster Ovary (CHO) cells are considered to be the most representative mammalian cell expression system. Compared with other protein expression systems, the cell is easy to form an engineering cell line, can stably express the target protein, and is beneficial to large-scale production. At present, P72 protein can be successfully expressed, but the expression yield is low, and the development of a genetic engineering subunit vaccine cannot be met.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a construction method of an African swine fever virus p72 protein stable expression cell line, a CHO cell line for stably expressing p72 recombinant protein and a p72 recombinant protein with higher purity are obtained by a prokaryotic expression and eukaryotic expression method; the invention also aims to provide a cell line for stably expressing the African swine fever virus p72 protein, which has strong stability and high activity.
One of the purposes of the invention is realized by adopting the following technical scheme:
a construction method of a cell line for stably expressing the p72 protein of African swine fever virus comprises the following steps:
1) constructing a vector: analyzing the signal peptide and enzyme cutting site information of the gene sequence of the African swine fever p72 protein, wherein the nucleotide sequence of the p72 protein is shown as SEQ.ID NO: 1 is shown in the specification; (ii) a A Kozac sequence shown in SEQ ID NO. 2 is added before an initiation codon ATG, and a mouse IgG kappa chain signal peptide sequence shown in SEQ ID NO. 3 is connected to the N end of the sequence; connecting a histidine tag to the C end of the peptide chain, connecting the fusion protein with the target protein, and adding 42 amino acids to the front end of the fusion protein, wherein the 42 amino acids can be used as purification tags for subsequent detection and purification in order to promote genome replication, and the nucleotide sequence of the target protein after sequence modification is shown as SEQ.ID NO: 4, respectively cloning the target protein to a prokaryotic expression vector and a eukaryotic expression vector;
2) prokaryotic expression: transforming the prokaryotic expression vector subjected to cloning in the step 1) into competent escherichia coli), selecting a single clone, inoculating the single clone into an LB liquid culture medium containing kanamycin, and culturing; then inoculating the culture into LB liquid culture medium containing kanamycin, and continuing to culture; then adding IPTG (isopropyl thiogalactoside) for culturing, centrifugally collecting precipitates, adding a solvent for resuspension, carrying out ultrasonic treatment in an ice bath, centrifuging, separating the precipitates from a supernatant, and respectively detecting whether the p72 recombinant protein is expressed or not to the precipitates and the supernatant to obtain an expression product;
purifying the expression product by using a protein purifier, balancing a chromatographic column, then loading, performing gradient elution on samples by using imidazole containing different concentrations respectively, and taking the purified samples to detect whether the p72 recombinant protein is purified;
3) eukaryotic expression: transfecting the cloned eukaryotic expression vector in the step 1) to cultured CHO cells in a liposome mode, culturing in a carbon dioxide incubator, observing the expression condition of p72 protein by a fluorescence microscope, cracking the cultured cells by using cell lysis solution, and detecting whether the p72 recombinant protein is expressed or not by using the cell lysis solution;
4) and (3) performance detection: resuspending the precipitate obtained in the step 2) after ultrasonic treatment in prokaryotic expression, mixing the precipitate with the solution obtained in the step 3) after eukaryotic expression cell lysis, then mixing the solution with an alkaline substance, spreading the mixture in an enzyme label plate for coating, carrying out incubation and sealing by using a PBST solution containing skimmed milk powder, selecting pig ASFV positive serum as a first antibody and rabbit anti-pig HRP as a second antibody to detect the two antigens, and checking the combination condition of the p72 recombinant protein and the ASFV antibody in the positive serum to obtain the African swine fever virus p72 protein stable expression cell line.
Further, in the step 1), the histidine tag body is a peptide section formed by stringing 6-10 histidines; the fusion protein is a flexible Linker containing 2-6 tetrads; the prokaryotic expression vector is pET-26b (+); the eukaryotic expression vector is pCMV-EGFP.
Further, in the step 2), the prokaryotic expression vector cloned in the step 1) is transformed into competent escherichia coli BL21(DE3), and a single clone is selected and inoculated into an LB liquid culture medium containing kanamycin, and is subjected to shaking culture at the rotating speed of 150-200 rpm for overnight; inoculating the overnight culture into LB liquid culture medium containing kanamycin at a ratio of 1:100, and performing shake culture at a rotation speed of 150-200 rpm for 4-5 h to OD 600 0.3 to 0.6; adding IPTG to a final concentration of 0.3-0.6 mM, and carrying out shake culture at 35-38 ℃ for 4-5 h; centrifuging at the rotating speed of 7000-8000 rpm, collecting the precipitate, resuspending the precipitate with a PBS solution, performing ice bath ultrasonic treatment (10s/10s) at 200-300W for 20-30 min, centrifuging at the rotating speed of 8000-10000 rpm, separating the precipitate and a supernatant, and performing western blotting on the precipitate and the supernatant respectively to detect whether the p72 recombinant protein is expressed or not to obtain an expression product.
Further, in step 2), the expression product is purified using a protein purifier, the HisTrap column is equilibrated using PB buffer solution having pH of 7.0 to 7.5, followed by loading, gradient elution is performed on samples containing 100mM, 200mM, 300mM, 400mM, and 500mM imidazole having pH of 7.5 to 8.0, respectively, and the purified samples are taken to perform western blotting to detect whether the p72 recombinant protein is purified.
Still further, in step 3), the culture steps of the CHO cells are: CHO cells were resuscitated in cell flasks using F12-K medium containing 10% fetal bovine serum, according to 1: 3 until the cell amount is 0.2-7 x 10 8 (ii) individual cells; the newly cultured CHO cells were cultured at (0.5-2). times.10 6 Inoculating 6-hole cell culture plates at the density of each cell, and culturing in a carbon dioxide incubator at 37 ℃ for 18-24 h.
Further, in the step 3), the cloned eukaryotic expression vector is transfected to cultured CHO cells in a liposome mode, the amount of transfected DNA in each hole is 2-5 mu g, the CHO cells are placed in a carbon dioxide incubator and cultured for 48-72 h at 35-38 ℃, the expression condition of p72 protein is observed through a fluorescence microscope, the cells cultured for 48h and 72h are cracked by cell lysate, and the solution obtained after cell cracking is subjected to western blotting to detect whether the p72 recombinant protein is expressed.
Further, in the step 4), the precipitate obtained in the step 2) after ultrasonic treatment in prokaryotic expression is resuspended, and the precipitate is mixed with the solution obtained in the step 3) after eukaryotic expression cell cracking and then with NaHCO with the pH value of 9-10 3 Mixing the solutions, paving the mixed solution into a 96-well enzyme label plate at a ratio of 50 ng/well for coating, coating overnight at 4 ℃, incubating and sealing at 37 ℃ by using a PBST solution containing 5% skimmed milk powder, selecting pig ASFV positive serum diluted by 200 times with PBST as a first antibody, selecting rabbit anti-pig HRP diluted by 10000 times with PBST as a second antibody for detecting the two antigens, and checking the combination condition of p72 recombinant protein and the ASFV antibody in the positive serum to obtain the African swine fever virus p72 protein stable expression cell line.
The second purpose of the invention is realized by adopting the following technical scheme:
an African swine fever virus p72 protein stable expression cell line is prepared by the construction method of the African swine fever virus p72 protein stable expression cell line.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the cell line construction method, after the p72 protein is subjected to sequence transformation to obtain recombinant protein, the recombinant protein is respectively cloned to a prokaryotic expression vector and an eukaryotic expression vector, and prokaryotic expression and eukaryotic expression are respectively carried out; wherein, in the process of prokaryotic expression, escherichia coli which stably expresses p72 recombinant protein and purified p72 recombinant protein can be obtained; after eukaryotic expression, a CHO cell strain capable of stably expressing p72 recombinant protein can be obtained. The construction method can be used for screening and obtaining the CHO cell strain expressing the African swine fever virus p72 protein within 2 months, and the cell line can still detect the exogenous recombinant protein when the cell line is transmitted to 20 generations under the pressure of no puromycin screening, and the success rate is 90 percent.
(2) The stable expression African swine fever virus p72 protein CHO cell line obtained by the construction method can still express p72 recombinant protein after continuous transmission for 15-20 generations.
(3) The expressed p72 recombinant protein can be used for researching antigens for ASFV serodiagnosis, and has very important significance for ASFV diagnosis and vaccine production.
Drawings
FIG. 1 is the identification chart of plasmid pET26b-p72 cut by BamH I and Xho I;
FIG. 2 is the identification chart of plasmid pCMV-EGFP-p72 digested by EcoR I and Bgl II;
FIG. 3 is a 48h fluorescence microscope observation image of eukaryotic expression vector transfected CHO cells;
FIG. 4 is a graph showing the identification of western blots expressing p72 protein by CHO cells;
FIG. 5 is a fluorescence microscope observation image of the selected monoclonal cell strain after eukaryotic expression.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A construction method of a cell line for stably expressing the p72 protein of African swine fever virus comprises the following steps:
1) constructing a vector: analyzing the signal peptide and enzyme cutting site information of the gene sequence of the African swine fever p72 protein, wherein the nucleotide sequence of the p72 protein is shown as SEQ.ID NO: 1 is shown in the specification; a Kozac sequence shown in SEQ ID NO. 2 is added before an ATG start codon, and a mouse IgG kappa chain signal peptide sequence shown in SEQ ID NO. 3 is connected to the N end of the sequence of the p72 protein; a histidine tag containing 6-10 histidines is connected to the C end of the peptide chain of the p72 protein, and 2-6 tetrads (G) 4 S) is connected with a target protein, and then 42 amino acids are added in front of the Linker, wherein the amino acids are used for promoting genome replication and can be used as a purification tag for subsequent detection and purification, and the nucleotide sequence of the target protein after sequence modification is shown as SEQ. ID NO: 4, the target proteinRespectively cloning to a prokaryotic expression vector pET-26b (+), and a eukaryotic expression vector pCMV-EGFP.
2) Prokaryotic expression: cloning p72 recombinant protein genes to a prokaryotic expression vector pET-26b (+) by an enzyme digestion connection method, converting the cloned prokaryotic expression vector into competent escherichia coli BL21(DE3) through PCR and sequencing verification, selecting a single clone, inoculating the single clone into an LB liquid culture medium containing kanamycin, and carrying out shaking culture at the rotating speed of 150rpm for overnight; the overnight culture was inoculated into LB liquid medium containing kanamycin at a ratio of 1:100, and shake-cultured at 200rpm for 4 hours to OD 600 Is 0.5; adding IPTG to a final concentration of 0.5mM, and culturing at 37 deg.C for 4h with shaking; centrifuging at 8000rpm to collect precipitate, resuspending the precipitate with PBS solution, performing 200W ice bath ultrasound (10s/10s) for 20min, centrifuging at 10000rpm, separating precipitate and supernatant, performing western blotting (western blots) on the precipitate and supernatant to detect whether p72 recombinant protein is expressed, and obtaining pET26b-p72 plasmid as shown in FIG. 1;
use of
Purifying the expression product by a protein purifier, balancing a HisTrap chromatographic column by using a PB buffer solution with the pH of 7.4, then loading, performing gradient elution on samples containing 100mM, 200mM, 300mM, 400mM and 500mM of imidazole with the pH of 8.0 respectively, and performing protein immunoblotting (western blots) on the purified samples to detect whether the p72 recombinant protein is purified or not; among them, the elution effect was the highest at an imidazole concentration of 300 mM.
3) Eukaryotic expression: cloning the p72 recombinant protein gene to a eukaryotic expression vector pCMV-EGFP by an enzyme digestion connection method, and verifying the correctness by PCR and sequencing, as shown in figure 2, so as to obtain a pCMV-EGFP-p72 plasmid; transfecting the cloned eukaryotic expression vector (pCMV-EGFP-p72) to cultured CHO cells in a liposome mode, wherein the amount of transfected DNA in each hole is 2 mu g, putting the cells into a carbon dioxide incubator, culturing for 48-72 h at 37 ℃, observing the expression condition of p72 protein by a fluorescence microscope, and observing a fluorescent microscope image after culturing for 48h as shown in figure 3; and the cells cultured for 48h and 72h are taken to be cracked by cell lysate, and the solution after cell cracking is taken to be subjected to Western blot detection, and the result is shown in figure 4, and the expression of the p72 recombinant protein can be detected. FIG. 5 shows the fluorescence microscopic observation image of the selected monoclonal cell line after eukaryotic expression.
4) And (3) performance detection: resuspending the precipitate obtained in step 2) after ultrasonic treatment, mixing with the solution obtained in step 3) after cell lysis, and mixing with NaHCO at pH 9.6 3 The solutions are mixed, and then the mixture is spread in a 96-well enzyme label plate for coating at 50 ng/well, the mixture is coated overnight at 4 ℃, a PBST solution containing 5% skimmed milk powder is used for incubation and sealing at 37 ℃, pig ASFV positive serum which is diluted by PBST by 200 times is selected as a first antibody, rabbit anti-pig HRP which is diluted by PBST by 10000 times is selected as a second antibody to detect the two antigens, and the combination of p72 recombinant protein and the ASFV antibody in the positive serum can be detected, so that the African swine fever virus p72 protein stable expression cell line is obtained.
Wherein, in the step 3), the culture step of the CHO cells comprises the following steps: CHO cells were resuscitated in cell flasks using F12-K medium containing 10% fetal bovine serum, according to 1: 3 until the cell mass is 5X 10 8 (ii) individual cells; freshly cultured CHO cells at 1X 10 6 The density of each cell is inoculated on a 6-hole cell culture plate, and the cell culture plate is placed in a carbon dioxide incubator at 37 ℃ for 24 hours.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
SEQUENCE LISTING
<110> Zhaoqing fen-center in Guangdong province laboratory of Lingnan modern agricultural science and technology
<120> African swine fever virus p72 protein stable expression cell line and construction method thereof
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1938
<212> DNA
<213> Artificial sequence
<400> 1
atggcatcag gaggagcttt ttgtcttatt gctaacgatg ggaaggccga caagattata 60
ttggcccaag acttgcttaa tagcaggatt tctaacatta aaaatgtgaa caaaagttat 120
gggaaacccg accccgaacc cactttgagt caaatcgaag aaacacattt ggttcatttt 180
aatgcgcatt ttaagcctta tgttccagta gggtttgaat acaataaagt acgcccgcat 240
acgggtaccc ccaccttggg aaacaagctt acctttggta ttccccagta cggagacttt 300
ttccatgata tggtgggcca ccatatattg ggtgcatgtc attcgtcctg gcaggatgct 360
ccgattcagg gcacggccca gatgggggcc catggtcagc ttcaaacgtt tcctcgcaac 420
ggatatgact gggacaacca aacaccttta gagggcgccg tttacacgct tgtagatccc 480
tttggaagac ctattgtacc cggcacaaag aatgcgtacc gaaacttggt ttactactgc 540
gaataccccg gagaacgact ttatgaaaac gtaagattcg atgtaaatgg aaattccctg 600
gacgaatata gttcggatgt cacaacgctt gtgcgcaaat tttgcatccc aggggataaa 660
atgactggat ataagcactt ggtcggccag gaggtatcgg tggagggaac tagtggccct 720
ctcctatgca acattcatga tttgcacaag ccgcaccaaa gcaaacctat tcttaccgat 780
gaaaatgata cgcagcgaac gtgcagccat accaacccga aattcctttc acaacatttt 840
cccgagaact ctcacaatat ccaaacagca ggtaaacaag atattactcc tattacggac 900
gcaacgtatc tggacataag acgtaatgtt cattacagct gtaatggacc tcaaacccct 960
aaatactatc agccccctct tgcgctctgg attaagctgc gcttttggtt taacgagaac 1020
gtgaaccttg ctattccctc ggtatccatt cccttcggcg agcgctttat caccataaag 1080
cttgcatcgc aaaaggattt ggtgaatgaa tttcctggac tttttatacg ccagtcgcgt 1140
tttatacctg gacgccccag tagacgcaat atacgcttta aaccatggtt tatcccagga 1200
gtcattaatg aaatctcgct cacgaataat gaactttaca tcaataacct gtttgtaacc 1260
cctgaaatac acaacctttt tgtaaaacgc gttcgatttt ccctgatacg tgtccataaa 1320
acgcaggtga cccacaccaa caataaccac cacgatgaaa aactaatgtc tgctcttaaa 1380
tggcccattg aatatatgtt tataggatta aaacctacct ggaacatctc cgatcaaaat 1440
cctcatcaac accgagattg gcacaagttc ggacatgttg ttaacgccat tatgcagcct 1500
actcaccacg cagagataag ctttcaggat agagatacag ctcttccaga cgcatgttca 1560
tctatatcgg atattagccc cgttacgtat ccgatcacat tacctattat taaaaacatt 1620
tccgtaactg ctcatggtat caatcttatc gataagtttc catcaaagtt ctgcagctct 1680
tacataccct tccactacgg aggcaatgca attaaaaccc ccgatgatcc gggtgcgatg 1740
atgattacct ttgctttgaa gccacgggag gaataccaac ccagtggtca tattaacgta 1800
tccagagcaa gagaatttta tattagttgg gacacggatt acgtggggtc tatcactacg 1860
gctgatcttg tggtatcggc atctgctatt aactttcttc ttcttcagaa cggttcagct 1920
gtgctgcgtt acagtacc 1938
<210> 2
<211> 6
<212> DNA
<213> Artificial sequence
<400> 2
gccacc 6
<210> 3
<211> 60
<212> DNA
<213> Artificial sequence
<400> 3
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60
<210> 4
<211> 2070
<212> DNA
<213> Artificial sequence
<400> 4
gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60
actggtatgg catcaggagg agctttttgt cttattgcta acgatgggaa ggccgacaag 120
attatattgg cccaagactt gcttaatagc aggatttcta acattaaaaa tgtgaacaaa 180
agttatggga aacccgaccc cgaacccact ttgagtcaaa tcgaagaaac acatttggtt 240
cattttaatg cgcattttaa gccttatgtt ccagtagggt ttgaatacaa taaagtacgc 300
ccgcatacgg gtacccccac cttgggaaac aagcttacct ttggtattcc ccagtacgga 360
gactttttcc atgatatggt gggccaccat atattgggtg catgtcattc gtcctggcag 420
gatgctccga ttcagggcac ggcccagatg ggggcccatg gtcagcttca aacgtttcct 480
cgcaacggat atgactggga caaccaaaca cctttagagg gcgccgttta cacgcttgta 540
gatccctttg gaagacctat tgtacccggc acaaagaatg cgtaccgaaa cttggtttac 600
tactgcgaat accccggaga acgactttat gaaaacgtaa gattcgatgt aaatggaaat 660
tccctggacg aatatagttc ggatgtcaca acgcttgtgc gcaaattttg catcccaggg 720
gataaaatga ctggatataa gcacttggtc ggccaggagg tatcggtgga gggaactagt 780
ggccctctcc tatgcaacat tcatgatttg cacaagccgc accaaagcaa acctattctt 840
accgatgaaa atgatacgca gcgaacgtgc agccatacca acccgaaatt cctttcacaa 900
cattttcccg agaactctca caatatccaa acagcaggta aacaagatat tactcctatt 960
acggacgcaa cgtatctgga cataagacgt aatgttcatt acagctgtaa tggacctcaa 1020
acccctaaat actatcagcc ccctcttgcg ctctggatta agctgcgctt ttggtttaac 1080
gagaacgtga accttgctat tccctcggta tccattccct tcggcgagcg ctttatcacc 1140
ataaagcttg catcgcaaaa ggatttggtg aatgaatttc ctggactttt tatacgccag 1200
tcgcgtttta tacctggacg ccccagtaga cgcaatatac gctttaaacc atggtttatc 1260
ccaggagtca ttaatgaaat ctcgctcacg aataatgaac tttacatcaa taacctgttt 1320
gtaacccctg aaatacacaa cctttttgta aaacgcgttc gattttccct gatacgtgtc 1380
cataaaacgc aggtgaccca caccaacaat aaccaccacg atgaaaaact aatgtctgct 1440
cttaaatggc ccattgaata tatgtttata ggattaaaac ctacctggaa catctccgat 1500
caaaatcctc atcaacaccg agattggcac aagttcggac atgttgttaa cgccattatg 1560
cagcctactc accacgcaga gataagcttt caggatagag atacagctct tccagacgca 1620
tgttcatcta tatcggatat tagccccgtt acgtatccga tcacattacc tattattaaa 1680
aacatttccg taactgctca tggtatcaat cttatcgata agtttccatc aaagttctgc 1740
agctcttaca tacccttcca ctacggaggc aatgcaatta aaacccccga tgatccgggt 1800
gcgatgatga ttacctttgc tttgaagcca cgggaggaat accaacccag tggtcatatt 1860
aacgtatcca gagcaagaga attttatatt agttgggaca cggattacgt ggggtctatc 1920
actacggctg atcttgtggt atcggcatct gctattaact ttcttcttct tcagaacggt 1980
tcagctgtgc tgcgttacag taccggtgga ggcggtagtg gcggaggcgg ttcaggcgga 2040
ggcggatctc accaccacca ccaccactaa 2070
Claims (8)
1. A construction method of a cell line for stably expressing the p72 protein of African swine fever virus is characterized by comprising the following steps:
1) constructing a vector: analyzing the signal peptide and enzyme cutting site information of the gene sequence of the African swine fever p72 protein, wherein the nucleotide sequence of the p72 protein is shown as SEQ.ID NO: 1 is shown in the specification; a Kozac sequence shown in SEQ ID NO. 2 is added before an initiation codon ATG, and a mouse IgG kappa chain signal peptide sequence shown in SEQ ID NO. 3 is connected to the N end of the sequence; connecting a histidine tag to the C end of the peptide chain, connecting the fusion protein with the target protein, adding a purification tag to the front end of the fusion protein, and finishing the sequence modification to obtain the nucleotide sequence of the target protein shown as SEQ. ID NO: 4, respectively cloning the target protein to a prokaryotic expression vector and a eukaryotic expression vector;
2) prokaryotic expression: transforming the prokaryotic expression vector subjected to cloning in the step 1) into competent escherichia coli, selecting a single clone, inoculating the single clone into an LB liquid culture medium containing kanamycin, and culturing; then inoculating the culture into LB liquid culture medium containing kanamycin, and continuing to culture; then adding IPTG (isopropyl thiogalactoside) for culturing, centrifugally collecting precipitates, adding a solvent for resuspension, carrying out ultrasonic treatment in an ice bath, centrifuging, separating the precipitates from a supernatant, and respectively detecting whether the p72 recombinant protein is expressed or not to the precipitates and the supernatant to obtain an expression product;
purifying the expression product by using a protein purifier, balancing a chromatographic column, then loading, performing gradient elution on samples by using imidazole containing different concentrations respectively, and taking the purified samples to detect whether the p72 recombinant protein is purified;
3) eukaryotic expression: transfecting the cloned eukaryotic expression vector in the step 1) to cultured CHO cells in a liposome mode, culturing in a carbon dioxide incubator, observing the expression condition of p72 protein by a fluorescence microscope, cracking the cultured cells by using cell lysis solution, and detecting whether the p72 recombinant protein is expressed or not by using the cell lysis solution;
4) and (3) performance detection: resuspending the precipitate obtained in the step 2) after ultrasonic treatment in prokaryotic expression, mixing the precipitate with the solution obtained in the step 3) after eukaryotic expression cell lysis, then mixing the solution with an alkaline substance, spreading the mixture in an enzyme label plate for coating, carrying out incubation and sealing by using a PBST solution containing skimmed milk powder, selecting pig ASFV positive serum as a first antibody and rabbit anti-pig HRP as a second antibody to detect the two antigens, and checking the combination condition of the p72 recombinant protein and the ASFV antibody in the positive serum to obtain the African swine fever virus p72 protein stable expression cell line.
2. The method for constructing the African swine fever virus p72 protein stable expression cell line according to claim 1, wherein in the step 1), the histidine tag is a peptide fragment consisting of 6-10 histidines; the fusion protein is a flexible Linker containing 2-6 quadruplets; the prokaryotic expression vector is pET-26b (+); the eukaryotic expression vector is pCMV-EGFP.
3. The method for constructing the stable expression cell line of the African swine fever virus p72 protein according to claim 1, wherein in the step 2), the prokaryotic expression vector cloned in the step 1) is transformed into competent Escherichia coli BL21(DE3), and a single clone is selected and inoculated into an LB liquid medium containing kanamycin, and is subjected to shaking culture at the rotating speed of 150-200 rpm for overnight; inoculating the overnight culture into LB liquid culture medium containing kanamycin at a ratio of 1:100, and performing shake culture at a rotation speed of 150-200 rpm for 4-5 h to OD 600 0.3 to 0.6; adding IPTG to a final concentration of 0.3-0.6 mM, and carrying out shake culture at 35-38 ℃ for 4-5 h; centrifuging at the rotating speed of 7000-8000 rpm, collecting the precipitate, resuspending the precipitate with a PBS solution, performing ice bath ultrasonic treatment (10s/10s) at 200-300W for 20-30 min, centrifuging at the rotating speed of 8000-10000 rpm, separating the precipitate and a supernatant, and performing western blotting on the precipitate and the supernatant respectively to detect whether the p72 recombinant protein is expressed or not to obtain an expression product.
4. The method for constructing the African swine fever virus p72 protein stable expression cell line according to claim 1 or 3, wherein in step 2), the expression product is purified by using a protein purifier, the HisTrap chromatographic column is equilibrated by using PB buffer solution with pH of 7.0-7.5, then the sample is loaded, the sample is subjected to gradient elution by using 100mM, 200mM, 300mM, 400mM and 500mM imidazole with pH of 7.5-8.0 respectively, and the purified sample is subjected to protein immunoblotting to detect whether the p72 recombinant protein is purified.
5. The method for constructing the stable African swine fever virus p72 protein expression cell line of claim 1, wherein in the step 3), the culture step of the CHO cells comprises: CHO cells were resuscitated in cell flasks using F12-K medium containing 10% fetal bovine serum, according to 1: 3 until the cell amount is 0.2-7 x 10 8 (ii) individual cells; the newly cultured CHO cells were cultured at (0.5-2). times.10 6 Inoculating 6-hole cell culture plates at the density of each cell, and culturing in a carbon dioxide incubator at 37 ℃ for 18-24 h.
6. The method for constructing the African swine fever virus p72 protein stable expression cell line according to claim 1 or 5, wherein in the step 3), the cloned eukaryotic expression vector is transfected into cultured CHO cells in a liposome manner, the amount of transfected DNA per well is 2-5 μ g, the cultured CHO cells are placed into a carbon dioxide incubator and cultured for 48-72 h at 35-38 ℃, the expression condition of the p72 protein is observed by a fluorescence microscope, the cells cultured for 48h and 72h are lysed by using a cell lysate, and a solution obtained after cell lysis is subjected to western blotting to detect whether the p72 recombinant protein is expressed.
7. The method for constructing the African swine fever virus p72 protein stable expression cell line of claim 1, wherein in the step 4), the precipitate obtained in the step 2) after ultrasonic treatment in prokaryotic expression is resuspended, and the pellet is lysed with the solution obtained in the step 3) after eukaryotic expression cells are lysed, and then the pellet is mixed with NaHCO with the pH value of 9-10 3 Mixing the solutions, paving the mixed solution into a 96-well enzyme label plate at a ratio of 50 ng/well for coating, coating overnight at 4 ℃, incubating and sealing at 37 ℃ by using a PBST solution containing 5% skimmed milk powder, selecting pig ASFV positive serum diluted by 200 times with PBST as a first antibody, selecting rabbit anti-pig HRP diluted by 10000 times with PBST as a second antibody for detecting the two antigens, and checking the combination condition of p72 recombinant protein and the ASFV antibody in the positive serum to obtain the African swine fever virus p72 protein stable expression cell line.
8. An African swine fever virus p72 protein stable expression cell line, characterized in that the cell line is prepared by the construction method of the African swine fever virus p72 protein stable expression cell line of any claim 1-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699860.3A CN115044612A (en) | 2022-06-20 | 2022-06-20 | African swine fever virus p72 protein stable expression cell line and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210699860.3A CN115044612A (en) | 2022-06-20 | 2022-06-20 | African swine fever virus p72 protein stable expression cell line and construction method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115044612A true CN115044612A (en) | 2022-09-13 |
Family
ID=83162562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210699860.3A Pending CN115044612A (en) | 2022-06-20 | 2022-06-20 | African swine fever virus p72 protein stable expression cell line and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115044612A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107746848A (en) * | 2017-11-03 | 2018-03-02 | 南京钟鼎生物技术有限公司 | Recombinate CSFV E 2 protein and its expression cell system, preparation method, application and CSFV subunit vaccine |
| CN111234036A (en) * | 2020-03-10 | 2020-06-05 | 天康生物(上海)有限公司 | African swine fever virus p72 fusion protein and preparation method and application thereof |
| KR20200143267A (en) * | 2019-06-14 | 2020-12-23 | 연세대학교 원주산학협력단 | p72 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof |
-
2022
- 2022-06-20 CN CN202210699860.3A patent/CN115044612A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107746848A (en) * | 2017-11-03 | 2018-03-02 | 南京钟鼎生物技术有限公司 | Recombinate CSFV E 2 protein and its expression cell system, preparation method, application and CSFV subunit vaccine |
| KR20200143267A (en) * | 2019-06-14 | 2020-12-23 | 연세대학교 원주산학협력단 | p72 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof |
| CN111234036A (en) * | 2020-03-10 | 2020-06-05 | 天康生物(上海)有限公司 | African swine fever virus p72 fusion protein and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李文傲: "非洲猪瘟病毒P72 蛋白单克隆抗体的制备", 中国优秀硕士学位论文全文数据库农业科技辑, no. 12, 15 December 2020 (2020-12-15), pages 14 - 18 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110078801B (en) | CHO cell strain for efficiently expressing African swine fever CD2V protein | |
| CN111234036B (en) | African swine fever virus p72 fusion protein and preparation method and application thereof | |
| CN112472801A (en) | DNA vaccine and subunit vaccine of African swine fever p30, p54, p72 and B602L, and preparation method and application thereof | |
| CN112574319B (en) | African swine fever virus P12 protein nanoparticle and preparation method and application thereof | |
| CN111607001A (en) | Recombinant soluble African swine fever virus p72 subunit fusion protein and preparation method and application thereof | |
| CN115160411A (en) | Screening, preparation and application of African swine fever virus dominant antigen | |
| CN113943354A (en) | Recombinant feline herpesvirus gB protein antigen and application thereof in antibody diagnosis and vaccine preparation | |
| CN113956362A (en) | Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation | |
| CN112521462B (en) | Horse infectious anemia virus p26-gp90 recombinant protein and preparation method and application thereof | |
| WO2021232800A1 (en) | Generic inert carrier escherichia coli and potential use thereof | |
| CN113832188A (en) | Novel coronavirus neutralizing antibody rapid detection kit based on fluorescent protein and application | |
| KR20210123234A (en) | Recombinant nucleocapsid protein for diagnosis and vaccine of COVID-19 and use thereof | |
| CN115044612A (en) | African swine fever virus p72 protein stable expression cell line and construction method thereof | |
| Wu et al. | Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB, gC, and gD proteins | |
| CN116804186A (en) | Anti-chicken infectious anemia virus monoclonal antibody hybridoma cell strain, monoclonal antibody, reagent or kit and application thereof | |
| CN113512098B (en) | Indirect ELISA (enzyme-Linked immuno sorbent assay) method for identifying swine fever virus and bovine viral diarrhea virus serum antibodies and application thereof | |
| CN111349637B (en) | Mycoplasma caprae and subspecies pneumoniae specific proteins and indirect ELISA kit | |
| CN115466317A (en) | Polyclonal antibody prepared based on PEDV ORF3 recombinant protein and established indirect ELISA detection method | |
| CN113588945A (en) | Novel coronavirus neutralizing antibody detection reagent and kit based on chemiluminescence | |
| CN113817027A (en) | Prokaryotic soluble expression method of bovine herpes virus type I gE protein extracellular region | |
| CN114487397A (en) | Indirect ELISA detection kit for detecting porcine delta coronavirus | |
| CN110724690A (en) | Cloning and prokaryotic expression method of goat SLAM gene | |
| CN115197961B (en) | African swine fever virus B602L recombinant protein stable expression cell line and construction method and application thereof | |
| CN118108838B (en) | Polyclonal antibody based on N protein of porcine delta coronavirus | |
| CN112521463B (en) | Ehrlichia canis MAP2-P30-gp19 recombinant protein and preparation method and application thereof |
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 |