CN108070599B - High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application - Google Patents
High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application Download PDFInfo
- Publication number
- CN108070599B CN108070599B CN201810086942.4A CN201810086942A CN108070599B CN 108070599 B CN108070599 B CN 108070599B CN 201810086942 A CN201810086942 A CN 201810086942A CN 108070599 B CN108070599 B CN 108070599B
- Authority
- CN
- China
- Prior art keywords
- recombinant
- porcine interleukin
- interleukin
- escherichia coli
- coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010074109 interleukin-22 Proteins 0.000 title claims abstract description 113
- 102100030703 Interleukin-22 Human genes 0.000 title claims abstract description 101
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 41
- 239000013612 plasmid Substances 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 239000013604 expression vector Substances 0.000 claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 238000000746 purification Methods 0.000 claims abstract description 9
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 37
- 239000006228 supernatant Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 14
- 238000012795 verification Methods 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 210000003000 inclusion body Anatomy 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 9
- 239000004202 carbamide Substances 0.000 claims description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 7
- 238000010276 construction Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 abstract description 13
- 241000894006 Bacteria Species 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 10
- 238000003259 recombinant expression Methods 0.000 abstract description 9
- 239000013598 vector Substances 0.000 abstract description 6
- 206010061126 Escherichia infection Diseases 0.000 abstract description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 4
- 208000020612 escherichia coli infection Diseases 0.000 abstract description 4
- 230000006909 anti-apoptosis Effects 0.000 abstract description 2
- 108010063738 Interleukins Proteins 0.000 abstract 1
- 102000015696 Interleukins Human genes 0.000 abstract 1
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 22
- 229930002954 deoxynivalenol Natural products 0.000 description 22
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 22
- 239000013592 cell lysate Substances 0.000 description 15
- 239000008055 phosphate buffer solution Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 10
- 230000006907 apoptotic process Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 7
- 238000004153 renaturation Methods 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 101000663658 Aptenodytes patagonicus Spheniscin-1 Proteins 0.000 description 6
- 241000282898 Sus scrofa Species 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- 102000004495 STAT3 Transcription Factor Human genes 0.000 description 5
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108010002069 Defensins Proteins 0.000 description 3
- 102000000541 Defensins Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000015728 Mucins Human genes 0.000 description 2
- 108010063954 Mucins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000008673 vomiting Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WWYNJERNGUHSAO-XUDSTZEESA-N (+)-Norgestrel Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](CC)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 WWYNJERNGUHSAO-XUDSTZEESA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 101150068065 Muc13 gene Proteins 0.000 description 1
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108091058544 REG family proteins Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102000013674 S-100 Human genes 0.000 description 1
- 108700021018 S100 Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 210000002203 alpha-beta t lymphocyte Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 1
- 208000030304 gastrointestinal bleeding Diseases 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000004964 innate lymphoid cell Anatomy 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 208000037817 intestinal injury Diseases 0.000 description 1
- 230000004673 intestinal mucosal barrier function Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229960004400 levonorgestrel Drugs 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- -1 trichothecene compound Chemical class 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种编码重组猪白介素22的基因。本发明还公开含有所述重组基因的重组表达载体、转基因细胞系统、转基因重组菌和宿主细胞。本发明还公开重组猪白介素22及其提取纯化方法。本发明还公开重组质粒pET‑32a（+）‑pIL‑22、重组菌株，重组蛋白在抗凋亡和抗大肠杆菌感染的应用。本发明构建的表达重组猪白介素22的载体pET‑32a（+）中含有His标签，从大肠杆菌中提取的重组猪白介素22蛋白与His标签相连，可通过His的柱子得到高度纯化的重组猪白介素22蛋白；重组猪白介素22蛋白表达产量通过Quantity One软件统计，纯度高达95.7%。
The invention discloses a gene encoding recombinant porcine interleukin 22. The invention also discloses a recombinant expression vector, a transgenic cell system, a transgenic recombinant bacteria and a host cell containing the recombinant gene. The invention also discloses the recombinant porcine interleukin 22 and its extraction and purification method. The invention also discloses the application of recombinant plasmid pET-32a(+)-pIL-22, recombinant strain and recombinant protein in anti-apoptosis and anti-Escherichia coli infection. The vector pET-32a(+) for expressing recombinant porcine interleukin 22 constructed by the present invention contains a His tag, the recombinant porcine interleukin 22 protein extracted from Escherichia coli is connected with the His tag, and a highly purified recombinant porcine interleukin can be obtained through the His column 22 protein; The expression yield of recombinant porcine interleukin 22 protein was calculated by Quantity One software, and the purity was as high as 95.7%.
Description
Technical Field
The invention relates to Escherichia coli expressing interleukin 22 of pig, it is a genetic engineering field of biotechnology; the method specifically comprises the following steps: the construction of a plasmid pET-32a (+) -pIL-22 for expressing the recombinant porcine interleukin 22, the extraction and purification of the expressed recombinant porcine interleukin 22 and the application of the recombinant expressed porcine interleukin 22 in the apoptosis induced by the Deoxynivalenol (DON) and the Escherichia coli infection resistance.
Background
1. Research progress of porcine interleukin 22
Porcine interleukin 22 is one of the interleukin 10 family members, which is mainly produced by the lymphoid system cell lines in innate and adaptive immunity, including α β T cells, γ δ T cells, natural killer cells and natural lymphocytes (ILCs), and macrophages, neutrophils can also produce interleukin 22 in addition to lymphoid tissues. The research reports that IL-22 has multiple physiological functions, can induce the expression of innate antibiotics (defensin, Reg family molecules and S100 protein) and the secretion of mucin (Muc1, Muc3, Muc10 and Muc13) and plays a functional barrier for intestinal mucosa, and meanwhile, IL-22 can jointly act with antibacterial defensin and mucosal immune antiviral cytokine IFN-lambda to improve the tissue barrier of IEC so as to relieve the infection of mucosal viruses. In recent years, the research on IL-22 mainly focuses on human sources and mouse sources, but the research on the porcine IL-22 is only rarely reported. It is not clear whether porcine interleukin 22 could be studied against apoptosis by activating the STAT3 signal and against e.
2. Application of porcine interleukin 22 in resisting apoptosis induced by Deoxynivalenol (DON) and resisting Escherichia coli infection
Deoxynivalenol (DON) is a trichothecene compound, secondary metabolites generated by fusarium exist in crops, food and feed, cause vomiting, diarrhea, gastrointestinal bleeding and the like of animals, seriously threaten the health of the animals, and pigs are the most sensitive animals to the DON, researches show that relatively low concentration (more than or equal to 50 mug/kg b.w.) can cause the vomiting of the pigs, and the DON can induce the enterocyte of the pigs to be apoptotic, so that the intestinal mucosa barrier is damaged, and the invasion of pathogenic microorganisms such as enteroviruses, clostridium and escherichia coli is promoted. Human or mouse interleukin 22 has been proved to induce the body to produce antibiotic substances such as innate antibiotics and mucin in human and mouse, thereby repairing chronic colitis and acute intestinal injury. It is not clear whether the porcine interleukin 22 can resist apoptosis induced by Deoxynivalenol (DON) and infection of Escherichia coli.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a gene for coding recombinant porcine interleukin 22.
The technical problem to be solved by the invention is to provide a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium containing the gene for coding the recombinant porcine interleukin 22.
The technical problem to be solved by the invention is to provide the gene for coding the recombinant porcine interleukin 22, the recombinant expression vector, the transgenic cell system or the application of the transgenic recombinant bacteria in the production of the recombinant porcine interleukin 22.
The technical problem to be solved by the invention is to provide the construction of the recombinant plasmid pET-32a (+) -pIL-22.
The technical problem to be solved by the invention is to provide an extraction and purification method of the recombinant porcine interleukin 22.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a gene for coding recombinant porcine interleukin 22, and the base sequence of the gene is shown as SEQ ID NO:1 is shown.
The invention also comprises a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium containing the gene for coding the recombinant porcine interleukin 22.
Wherein the recombinant expression vector is obtained by inserting the gene into an escherichia coli expression vector to obtain the expression vector containing the gene of the recombinant porcine interleukin 22.
Wherein, the Escherichia coli expression vector is pET-32a (+).
The invention also comprises a transgenic recombinant bacterium, wherein the recombinant bacterium is obtained by introducing the recombinant expression vector into escherichia coli and screening.
The invention also comprises a host cell which is obtained by introducing the recombinant expression vector into Escherichia coli.
The invention also comprises a recombinant protein which is obtained by the expression of the transgenic recombinant bacteria or the host cells.
The gene for coding the recombinant porcine interleukin 22, the recombinant expression vector, the transgenic cell system or the transgenic recombinant bacterium are applied to the production of the recombinant porcine interleukin 22.
The invention also discloses an extraction and purification method of the recombinant porcine interleukin 22, which comprises the following steps:
1) obtaining a recombinant porcine interleukin 22 gene;
2) constructing a recombinant plasmid pET-32a (+) -pIL-22;
3) expressing the recombinant escherichia coli;
4) and (3) extracting and purifying the recombinant porcine interleukin 22.
The recombinant porcine interleukin 22 gene is obtained by performing codon optimization on the porcine interleukin 22 gene, adding homologous arms of pET-32a (+) containing enzyme cutting sites Kpn I and Hind III at two ends of a gene sequence and artificially synthesizing;
the construction method of the recombinant plasmid pET-32a (+) -pIL-22 comprises the following steps: linearizing pET-32a (+) plasmid by using restriction enzymes Kpn I and Hind III, and connecting the linearized pET-32a (+) plasmid with the recombinant porcine interleukin 22 gene fragment synthesized in the step 1) to obtain pET-32a (+) -pIL-22 plasmid. The carrier pET-32a (+) for expressing the porcine interleukin 22 constructed by the invention contains a His label, the porcine interleukin 22 is connected with the His label, and the highly purified porcine interleukin 22 can be obtained through a His column.
Wherein, the expression method of the recombinant Escherichia coli comprises the following steps: the recombinant plasmid pET-32a (+) -pIL-22 is introduced into E.coli BL21(DE3) for expression, and positive recombinant Escherichia coli is obtained through colony PCR verification.
Wherein, the steps of extracting and purifying the recombinant porcine interleukin 22 are as follows: inducing and expressing the recombinant Escherichia coli with positive verification to obtain thalli, adding PBS (phosphate buffer solution) into the thalli for resuspension and centrifugation, carrying out ultrasonic crushing on the resuspended liquid, centrifuging the crushed bacterial liquid, collecting supernatant and precipitate for SDS-PAGE verification, finding that target protein exists in a large amount in an inclusion body and hardly exists in the supernatant, carrying out urea denaturation on the inclusion body precipitate, then carrying out gradient dialysis renaturation, collecting liquid after renaturation, passing through a His column, and purifying to obtain the porcine interleukin 22.
The recombinant expression vector, the transgenic recombinant bacteria, the host cell and the recombinant protein are applied to anti-apoptosis and anti-Escherichia coli infection.
Has the advantages that: compared with the prior art, the invention has the following characteristics and advantages:
1. the invention optimizes the gene sequence of the porcine interleukin 22 by adopting a codon optimization technology, and is beneficial to the recombinant gene porcine interleukin 22 to express the recombinant protein which is close to the natural conformation more possibly.
2. The plasmid pET-32a (+) -pIL-22 constructed in the experiment is the first plasmid which takes pET-32a (+) as a vector and can efficiently express the porcine interleukin 22 at home and abroad at present.
3. The invention successfully constructs plasmid pET-32a (+) -pIL-22 for expressing the porcine interleukin 22 and converts the plasmid into Escherichia coli E.coli BL21(DE 3). SDS-PAGE verifies that the porcine interleukin 22 can be successfully expressed in E.coli BL21(DE3), and highly purified porcine interleukin 22 can be obtained through His tag.
4. The invention adopts an optimized dialysis renaturation method to ensure that the inclusion body protein stably exists in a soluble state.
5. The porcine interleukin 22 expressed by the recombinant escherichia coli efficiently expressed by the invention can resist Deoxynivalenol (DON) induced apoptosis and can resist the adhesion of escherichia coli ETEC K88 to intestinal epithelial cells, and the expressed porcine interleukin 22 provides a new way for maintaining intestinal mucosal barriers and protecting organisms from being damaged by pathogenic microorganisms. Therefore, the application is expected to provide a new method and thought for the porcine interleukin 22 in the aspects of apoptosis resistance and infection resistance.
Drawings
FIG. 1: the plasmid pET-32a (+) -pIL-22 enzyme digestion verification electrophoretogram of the expression recombination pig interleukin 22; lane 1: m represents 5000bp DNA Marker; lane 2: representing plasmid pET-32a (+) -pIL-22; lane 3: representing the product of restriction enzyme digestion of plasmid pET-32a (+) -pIL-22 (restriction enzymes Kpn I and Hind III are used for digestion, and the restriction enzyme digestion fragments are respectively 5847bp of vector skeleton and 477bp of target fragment from top to bottom);
FIG. 2: schematic diagram of the constructed vector plasmid pET-32a (+) -pIL-22;
FIG. 3: recombinant Escherichia coli E.coli BL21(DE3) colony PCR verification electrophoretogram transferred into vector plasmid pET-32a (+) -pIL-22; lane 1: m represents a 15000bp DNA Marker, lane 2: negative control, lane: 3-10 represents a product after colony PCR (the target band size is 477 bp);
FIG. 4: SDS-PAGE electrophoresis picture of the recombinant porcine interleukin 22; lane 1: a protein Marker; lane 2: BSA (1. mu.g); lane 3: BSA (2 μ g); lane 4: (ii) uninduced cell lysate; lane 5: cell lysate under induction at 15 ℃ for 16 h; lane 6: cell lysate under induction at 37 ℃ for 4 h; lane 7: uninduced cell lysate supernatant; lane 8: (ii) uninduced cell lysate precipitation; lane 9: cell lysate supernatant induced at 15 ℃ for 16 h; lane 10: precipitating cell lysate under the induction of 16h at 15 ℃; lane 11: cell lysate supernatant induced at 37 ℃ for 4 h; lane 12: cell lysate precipitation induced at 37 ℃ for 4 h;
FIG. 5: western blot of recombinant porcine interleukin 22: lane 1: western blot Marker; lane 2: cell lysate under induction at 15 ℃ for 16 h; lane 3: cell lysate under induction at 37 ℃ for 4 h; lane 4: cell lysate supernatant induced at 15 ℃ for 16 h; lane 5: precipitating cell lysate under the induction of 16h at 15 ℃; lane 6: cell lysate supernatant induced at 37 ℃ for 4 h; lane 7: cell lysate precipitation induced at 37 ℃ for 4 h;
FIG. 6: SDS-PAGE electrophoresis chart of purified product verification of porcine interleukin 22 expressed by recombinant Escherichia coli E.coli BL21(DE3) transferred into carrier plasmid pET-32a (+) -pIL-22; lane 1: recombinant porcine interleukin 22 (purity 72.73%) after inclusion body denaturation; lane 2: recombinant porcine interleukin 22 (purity 77.65%) after renaturation; lane 3: ) The purified porcine interleukin 22 (purity 95.7%) of the His label Ni column; lane 4: a protein Marker;
FIG. 7: detecting result graphs and statistical graphs of the activation STAT3 of the expressed recombinant porcine interleukin 22 on IPEC-J2 cells by Western blot;
FIG. 8: flow detection of the result graph of the apoptosis of the expressed recombinant porcine interleukin 22 induced by Deoxynivalenol (DON);
FIG. 9: counting the result graph of the adhesion of the recombinant porcine interleukin 22 anti-Escherichia coli ETECK88 to the intestinal epithelial cells by a colony counting method;
FIG. 10: and (3) detecting the influence of the expressed recombinant porcine interleukin 22 on the expression quantity of the porcine defensin pBD-1 by real-time fluorescent quantitative PCR.
Detailed Description
The invention is further illustrated by the following specific examples and figures. The methods used in the following examples are conventional methods unless otherwise specified. The specific materials and reagents involved are as follows:
coli BL21(DE3) competent cells were purchased from norgestrel biotechnology ltd; pET-32a (+) plasmid was purchased from Life Technologies;
reagent: the Agarose Gel DNA Purification Kit, restriction enzyme, reverse transcription Kit were purchased from Takara bioengineering (Dalian) Co., Ltd; ampicillin was purchased from aeronautics bioengineering, Guangzhou, Inc. (Omega Bio-Tek agency);
II One Step Cloning Kit site-directed Cloning Kit was purchased from Nanjing Novozam Biotechnology Ltd.
EXAMPLE 1 construction of recombinant plasmid pET-32a (+) -pIL-22
1. Obtaining recombinant porcine interleukin 22 gene fragment
OptimumGene was used with reference to published gene sequences (GenBank accession: KX588234.1)TMGene design software, according to the preference of E.coli BL21 host, removing rare codons (AGA, GGA, CCC, CTA) of E.coli, adjusting the GC content of the sequence, optimizing the codons, designing the recombinant porcine interleukin 22 gene sequence as SEQ ID NO:1, and then adding homologous arms CAGCCCAGATCTGG of pET-32a (+) vector linearized by KpnI and Hind III enzymes into the two ends of the SEQ ID NO:1 gene sequenceGTACC(SEQ ID NO:2) and CGAGTGCGGCCGCAAGCTT(SEQ ID NO:3) (the restriction enzyme cleavage site is underlined), and the sequence was artificially synthesized (by Nanjing Kingsrey Biotech) to obtain a target fragment of the recombinant porcine interleukin 22 gene.
2. Construction of recombinant plasmid pET-32a (+) -pIL-22
The pET-32a (+) plasmid was linearized with the restriction enzymes Kpn I and Hind III. The reaction conditions were 2. mu.L each of Kpn I and Hind III, 10 XBuffer 5. mu.L, pET-32a (+) plasmidmu.L (total amount: 1. mu.g), 36. mu.L of ultrapure water, digested at 37 ℃ for 2 hours, and the digested product was recovered to obtain a linearized pET-32a (+) plasmid. The linearized pET-32a (+) plasmid and the fragment of the artificially synthesized recombinant porcine interleukin 22 gene are connected according to a connecting kit (
II One Step Cloning Kit purchased from Nykeda Biotechnology Ltd) to obtain plasmid pET-32a (+) -pIL-22, and the restriction enzymes Kpn I and Hind III were cut to verify the correctness, as shown in FIG. 1. The sequence obtained by the verification of the restriction products of the endonucleases Kpn I and Hind III is compared with the sequence SEQ ID NO. 1 for codon optimization by BLAST and DNAstar, and the result shows that the cloned recombinant gene sequence of the porcine interleukin 22 is completely consistent with the sequence SEQ ID NO. 1. The resulting recombinant plasmid pET-32a (+) -pIL-22 has a structure as shown in FIG. 2.
Example 2 verification of recombinant E.coli BL21(DE3) expressing porcine interleukin 22
mu.L (200 ng/. mu.L) of the recombinant plasmid pET-32a (+) -pIL-22 was added to 50. mu.L of E.coli BL21(DE3) chemically transformed competent cells, and the cells were placed on ice for 30min with no aspiration and a few flicks. And (3) performing heat shock at 42 ℃ for 90s in a water bath kettle, incubating on ice for 5min, adding 900 mu L of SOC culture medium, slightly reversing and uniformly mixing, putting the mixture into a 37 ℃ constant-temperature incubator for resuscitation for 10min, putting the recovered bacterial liquid into a 37 ℃ shaking table, culturing at 150rpm for 40min, taking out the bacterial liquid, centrifuging at 5000rpm for 5min, discarding the supernatant, taking 100 mu L of bacterial liquid, coating the bacterial liquid on an ampicillin resistant solid basal medium LB plate, and allowing positive bacterial colonies to be visible for 12-16 h. Colonies were picked for verification, as shown in fig. 3, 2-9 are colonies for PCR verification, the results are all positive, 1 is negative control, and the PCR verification primers are as follows:
P1：CCCAGATCTGGGTACCATGGTCCCG(SEQ ID NO:4)
P2：CGCCTTTAATACGACATTGGGACAGTT(SEQ ID NO:5)
the above primers were synthesized by Nanjing Kingsrei Biotech.
Example 3 extraction and purification of recombinant porcine interleukin 22 expressed by recombinant E.coli BL21(DE3)
Positive colonies of recombinant E.coli BL21(DE3) obtained in example 2 were picked and cultured overnight in LB medium containing ampicillin (50. mu.g/mL). Inoculating the overnight cultured bacterial liquid into a new LB culture medium in a ratio of 1:100 for culture, adding IPTG (isopropyl-beta-thiogalactoside) to induce the expression of the porcine interleukin-22 protein in the recombinant escherichia coli when the OD (acyl-D) value of the bacterial liquid reaches 0.5, and performing induced culture for 16h at 15 ℃ and 4h at 37 ℃. 8000rpm, 10min, centrifuging and collecting the thallus. PBS was added to resuspend the collected cells by centrifugation, and the resuspension solution was sonicated. After disruption, 14000rpm, 30min, centrifugation. Collecting supernatant and precipitate, performing SDS-PAGE and Western blot verification, wherein the result shows that a large amount of target protein exists in the inclusion body, the supernatant is almost not existed, modifying the collected inclusion body, performing dialysis renaturation (dialysis renaturation solution: 50mM Tris-HCl, 300mM NaCl, 0.5% TritonX-100, 0.5M arginine, 3mM reduced glutathione, 0.6mM oxidized glutathione, 20mM imidazole and urea with different concentrations), performing gradient dialysis on the modified target protein in the solution containing 8M urea, 6M urea, 4M urea, 2M urea, 1M urea, 0.5M urea and PBS, finally existing in a meltable state, passing the liquid obtained by dialysis renaturation through a His column, and purifying and extracting to obtain the recombinant porcine interleukin 22. The protein concentration of the porcine interleukin 22 is measured by a BCA method, the protein concentration obtained after purification is 8mg/ml, and the total amount of the porcine interleukin 22 which can be induced, expressed and purified by 100ml of bacterial liquid is 5 mg. SDS-PAGE electrophoresis verifies that the target protein porcine interleukin 22 exists, and as shown in figure 6, a Lane 1 is an inclusion body sediment of Escherichia coli E.coli BL21(DE3) transformed with pET-32a (+) -pIL-22 plasmid; lane 2 is porcine interleukin 22 renatured after denaturation of inclusion bodies; and the lane 3 is the pig interleukin 22 finally obtained by Ni column purification, the molecular weight of the purified pig interleukin 22 with the His label is 35.6kd, the molecular weight is consistent with the results of SDS-PAGE and Western blot, and the purity of the purified pig interleukin 22 is up to 95.7 percent through quantitative One software gray scale analysis.
Example 4 detection of STAT3 phosphorylation level 24h after recombinant porcine interleukin 22 acts on IPEC cells by Western blot
The IPEC-J2 cells with good recovery state (purchased from)Guangzhou Jinie Europe Biotech Co., Ltd.) were plated on 6-well cell culture plates at 2.5X 106A hole. Blank control group: add 2. mu.l of PBS per well (0.01M, pH 7.2); porcine interleukin 22 treatment group: one group was supplemented with 2. mu.l of the porcine interleukin 22 purified in example 3 (10ng/ml) and the other group with 2. mu.l of the porcine interleukin 22 purified in example 3 (100 ng/ml); porcine interleukin 22 and IPEC-J2 cells 5% CO at 37 ℃2After the incubator is used for 24 hours, the supernatant is discarded, PBS is added for washing for 3 times, 200 mu l of protein lysate (protein lysate RIPA: protease inhibitor PMSF is 100: 1) is added into each hole for collecting protein samples, 5xSDS-buffer protein buffer is added for heating and denaturation for 10min at 100 ℃, and then the result of Western blot shows that compared with a blank control group, when the concentration of the porcine interleukin 22 treatment group is 10ng/ml and 100ng/ml, the STAT3 phosphorylation level can be obviously increased by acting IPEC-J2 cells for 24 hours, and the STAT3 phosphorylation level is higher at 100 ng/ml. (see fig. 7).
Example 5: flow detection of expressed porcine interleukin 22 in anti-Deoxynivalenol (DON) induced apoptosis
The recovered IPEC-J2 cells (purchased from GmbH, GmbH) with good recovery status were plated in 6-well cell culture plates at 2.5X 106Per well. Blank control group: add 10. mu.L of 0.5% DMSO per well; DON treatment group: adding 10 μ l of deoxynivalenol (25 μ M) per well; IL-22 treatment group: add 2. mu.l of the porcine interleukin 22 purified in example 3 (100 ng/ml); after 24 hours of incubation at 37 ℃ in a 5% CO2 incubator, 2. mu.l of PBS (0.01M, pH 7.2) was added to the blank control group, and 2. mu.l of the porcine interleukin 22(100ng/ml) purified in example 3 was added to the DON treatment group for further 24 hours (IL-22 repair group), cell supernatants were collected, 200. mu.l/well of 0.25% trypsin (Vitrent Biotech Co., Ltd., product No. 325-040-EL) without EDTA was added thereto, the cells were digested and then discarded, the cells were washed with 0.01M PBS at pH7.2 for 1 time, and the collected supernatants and the digested cells were centrifuged at 1300rpm for 10min, followed by the operation according to the instructions of the apoptosis kit (Nanjing Nozavir Biotech Co., product Ltd., product No. A211-01). Adding 1ml binding buffer to wash the cells for 1 time, 1300rpm, centrifuging for 10min, discarding the cell supernatant, adding 100 μ l bind to the cell precipitateBuffer, gently resuspend cells with finger flick. Adding 10 μ l Annexin V-FITC, gently blowing and mixing, keeping away from light at 37 ℃ for 15min, washing with Binding buffer for 1-2 times, adding 500 μ l Binding buffer to resuspend cells, and finally measuring with a flow cytometer, and adding 5 μ l propidium iodide PI as nucleic acid dye before measurement. The experimental results show that: the number of apoptotic cells was counted, 18.6% in the blank control group, 17.11% in the IL-22 treatment group, 66.9 in the Deoxynivalenol (DON) treatment group, and 39.5% in the IL-22 repair group and the Deoxynivalenol (DON) induced apoptosis (FIG. 8).
Example 6: effect of recombinant porcine interleukin 22 on adhesion of escherichia coli resistant ETEC K88 to intestinal epithelial cells
Respectively paving recovered IPEC-J2 cells in a good state into 12-hole cell culture plates, rinsing the cells in each hole for 2 times by using ImL sterilized PBS when the cell fusion rate reaches about 90%, adding 2mL of a minimal medium without antibiotics, and adding a blank control group: add 2. mu.l PBS per well; 3 treatment groups: adding 2 μ l each of the purified porcine interleukin 22 of example 3 at concentrations of 10ng/ml and 100ng/ml for 12h, and adding 10 of the purified porcine interleukin 22 to the blank control group and the experimental group respectively8CFU E.coli ETECK88 (present by veterinarian of farm institute of Jiangsu province) was incubated in cell culture box for 2.5h, three wells (3 treatment groups were obtained: ETECK88, 10ng/ml IL-22+ ETECK88, and 100ng/ml IL-22+ ETECK88, respectively); rinsing the cells of each treatment group with a culture medium for 3 times, placing 0.5% TritonX-100200 μ l in a cell culture box, and incubating for 8min to lyse the cells; adding 250 mul of sterile double distilled water, repeatedly blowing, sucking out the sample suspension for multiple times, and performing 10 times of-7And 10-6After dilution by multiple times, coating an agar plate, culturing for 18-24 h in a constant-temperature bacteria incubator at 37 ℃, and counting the number of colonies. The experimental results show that: the porcine interleukin 22 can effectively reduce the adhesion of the Escherichia coli to intestinal epithelial cells at 10ng/ml and 100 ng/ml. (. P)<0.01) (see FIG. 9)
Example 7: the expression quantity of the porcine defensin can be improved by extracting the recombinant porcine interleukin 22 through real-time fluorescent quantitative PCR detection
Respectively spreading IPEC-J2 cells in good recovery state into 6-hole cell culture platesThe fusion rate of the cell in the hole reaches about 90 percent, 2.5 multiplied by 106One well, rinsed 2 times with 1mL of sterilized PBS, and 2mL of minimal medium without antibiotics added; blank control group: add 2. mu.l PBS per well; treatment group: IL-22 treatment group added with 2 u l 100ng/ml of porcine interleukin 22 purified in example 3 for 12h, Escherichia coli ETEC K88 (the gift from veterinarian of Jiangsu province farm institute) group added with 2 u l PBS; after 12h, 2.5X 10 additions were made to the E.coli ETEC K88 group and IL-22 treated group8Putting CFU ETECK88 into a cell culture box to incubate for 2.5h to respectively obtain an Escherichia coli ETEC K88 group and an IL-22+ ETEC K88 group, then rinsing cells of each treatment group for 3 times by using a culture medium, adding 1ml of RNA extraction reagent TRIzol into each hole to extract RNA of different treatment groups, obtaining cDNA after reverse transcription, and then carrying out real-time fluorescence quantitative PCR, wherein the real-time fluorescence quantitative PCR reaction system is 20 mu L, and the reaction system is as follows: SYBR 10. mu.l, Rox-I0.4. mu.l, cDNA 2. mu.l, forward primer P3(SEQ ID NO:6) 0.4. mu.l, reverse primer P4(SEQ ID NO:7) 0.4. mu.l and water 6.8. mu.l. (the real-time fluorescent quantitative PCR kit is Nanjing NuoZan Biotechnology Co., Ltd., product number: Q111-02), and the PCR reaction conditions are as follows: the relative expression level of swine alexin pBD-1 in each treatment group was quantitatively determined by pre-denaturation at 95 ℃ for 5min, at 95 ℃ for 10sec, at 60 ℃ for 30sec, for 30 cycles, at 95 ℃ for 10sec, at 60 ℃ for 1min, and at 95 ℃ for 15 sec. The experimental result shows that compared with a blank control group, the recombinant porcine interleukin 22 can up-regulate the expression of pBD-1, and meanwhile, the recombinant porcine interleukin 22 can improve the down regulation of Escherichia coli ETEC K88 to pBD-1. (. P)<0.01，*P<0.5) (see FIG. 10)
The foregoing is illustrative of selected embodiments of the present invention and is not intended to be exhaustive or to limit all embodiments to the precise form disclosed. It will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit of the invention, and these changes and modifications also fall within the scope of the invention.
Sequence listing
<110> Nanjing university of agriculture
<120> high-efficiency expression of recombinant porcine interleukin 22 in escherichia coli and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 477
<212> DNA
<213> recombinant porcine interleukin 22(Artificial Sequence)
<400> 1
atggtcccga ttacgcatca ctgcaaactg gatcagagca atttccaaca accgtatatt 60
accaaccgta cctttacgct ggcgcaggaa gcctcactgg cagataacaa tacggacgtt 120
cgcctgatcg gcaacaacct gtttcagggt gtcaatcaaa tgcgtgaacg ctgctatctg 180
gtgaaacagg ttctgaactt caccctggaa gaagtgctgt ttccgaattc ggatcgtttc 240
catccgtaca tgcaggaagt tgcgagtttt ctggacagcc tgtctaaaaa actgtcccaa 300
tgtcgtatta aaggcgatga ccagcacatc caacgcaacg tcaacaattt caaagatacg 360
gtgaaaaaac tgggcgaatc tggtgaaatt aaagtcatcg gtgaactgta cctgctgttt 420
atggctctga aaaatgaatg tacgctgccg ggtcactcgt ggaaaatgga caattaa 477
<210> 2
<211> 19
<212> DNA
<213> upstream homology arm (Artificial Sequence)
<400> 2
cagcccagat ctgggtacc 19
<210> 3
<211> 19
<212> DNA
<213> downstream homology arm (Artificial Sequence)
<400> 3
cgagtgcggc cgcaagctt 19
<210> 4
<211> 25
<212> DNA
<213> PCR verified primer P1(Artificial Sequence)
<400> 4
cccagatctg ggtaccatgg tcccg 25
<210> 5
<211> 27
<212> DNA
<213> PCR verified primer P2 (Artificial Sequence)
<400> 5
cgcctttaat acgacattgg gacagtt 27
<210> 6
<211> 20
<212> DNA
<213> upstream primer P3(Artificial Sequence) of pBD-1
<400> 6
<210> 7
<211> 18
<212> DNA
<213> downstream primer P4(Artificial Sequence) of pBD-1
<400> 7
ggtgccgatc tgtttcat 18
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810086942.4A CN108070599B (en) | 2018-01-30 | 2018-01-30 | High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810086942.4A CN108070599B (en) | 2018-01-30 | 2018-01-30 | High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108070599A CN108070599A (en) | 2018-05-25 |
| CN108070599B true CN108070599B (en) | 2021-07-02 |
Family
ID=62156927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810086942.4A Active CN108070599B (en) | 2018-01-30 | 2018-01-30 | High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108070599B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866145B (en) * | 2018-06-01 | 2022-03-08 | 河南德邻生物制品有限公司 | Detection steps and application of interaction between Saccharomyces cerevisiae and DON to reduce apoptosis of IPEC-J2 cells |
| CN111346218A (en) * | 2018-12-24 | 2020-06-30 | 四川华德生物工程有限公司 | Application of recombinant porcine interleukin 22 in preparation of medicament for improving production performance of pigs |
| CN110746501B (en) * | 2019-11-11 | 2021-08-20 | 南京农业大学 | Use of porcine interleukin 11 in resisting porcine epidemic diarrhea virus infection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103182072A (en) * | 2011-12-27 | 2013-07-03 | 健能隆医药技术（上海）有限公司 | Application of interleukin-22 in treating and preventing nerve injuries and neurodegeneration diseases |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7226591B2 (en) * | 2000-05-22 | 2007-06-05 | Genentech, Inc. | Interleukin-22 polypeptides, nucleic acids encoding the same and methods for the treatment of pancreatic disorders |
| AU2016259423B2 (en) * | 2007-11-07 | 2018-11-08 | Genentech, Inc. | Compositions and methods for treatment of microbial disorders |
| CN106146668A (en) * | 2015-04-01 | 2016-11-23 | 中国人民解放军第二军医大学 | The preparation method and its usage of long-acting interleukin II 2 |
-
2018
- 2018-01-30 CN CN201810086942.4A patent/CN108070599B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103182072A (en) * | 2011-12-27 | 2013-07-03 | 健能隆医药技术（上海）有限公司 | Application of interleukin-22 in treating and preventing nerve injuries and neurodegeneration diseases |
Non-Patent Citations (1)
| Title |
|---|
| 猪IL-22的原核表达及其活性研究;应兰等;《中国预防兽医学报》;20160615;第38卷(第06期);第489-492页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108070599A (en) | 2018-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108070599B (en) | High-efficiency expression of recombinant porcine interleukin-22 in Escherichia coli and its application | |
| CN116355887B (en) | A streptococcal prophage lytic enzyme lys733 and its application | |
| CN104593346A (en) | Endolysin from vibrio parahaemolyticus phage and application of endolysin | |
| CN111378660B (en) | A kind of sgRNA targeting tetracycline resistance gene tetA and its knockout vector, vector construction method and application | |
| CN111856006A (en) | Application of Mycoplasma bovis secreted protein MbovP274 | |
| CN111850002A (en) | Application of Mycoplasma bovis secreted protein MbovP570 | |
| CN109897861B (en) | Portunus trituberculatus chitinase gene, recombinant expression protein and application thereof | |
| CN108264548B (en) | Mandarin fish gamma interferon-related factor and its recombinant protein and application | |
| CN108148852B (en) | A kind of alginate lyase SHA-6 gene and application | |
| CN114657113A (en) | A kind of recombinant bacteria expressing totipotent nuclease and its application | |
| CN102492714A (en) | Method for preparing recombinant Chinese rural dog interferon-alpha | |
| CN103602684B (en) | Enhanced subsample gene capable of improving expression of foreign protein and application thereof | |
| CN111286464B (en) | An engineering bacterium expressing chitinase and its application in plant growth promotion | |
| US20230303651A1 (en) | Method for preparing a porcine-derived interferon-delta 5 and application of porcine-derived interferon-delta 5 | |
| CN112480227A (en) | Protein for improving pathogenic bacterium resistance of sturgeon and preparation method and application thereof | |
| CN111733178A (en) | Recombinant expression vector for improving soluble expression quantity of disease course related protein of astragalus mongholicus | |
| CN106632654B (en) | An optimized pIFN-γ peptide chain and its application in improving the production and activity of porcine IFN-γ secreted and expressed by Pichia pastoris | |
| CN113430220A (en) | Synthesis method, construction method and application of genetic engineering bacteria for expressing soluble feline omega interferon | |
| CN110437328B (en) | A kind of porcine interferon alpha mutant YNS and its preparation method and application | |
| CN104789513B (en) | A kind of coli strain for preparing bioactive peptide | |
| CN113355341A (en) | Fusion tag-based chitosanase expression promoting method and recombinant fusion chitosanase | |
| CN111154792A (en) | Preparation and application of cryptosporidium protein kinase 660C-terminal protein | |
| CN114292836A (en) | A kind of lyase for endocutting Salmonella phage, its encoding gene and its preparation method and application | |
| CN106749609B (en) | Chicken alpha interferon and preparation method thereof | |
| CN119709805A (en) | Method for efficiently expressing panda DNAJC12 gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |