CN103232545B - A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method - Google Patents
A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method Download PDFInfo
- Publication number
- CN103232545B CN103232545B CN201310182855.6A CN201310182855A CN103232545B CN 103232545 B CN103232545 B CN 103232545B CN 201310182855 A CN201310182855 A CN 201310182855A CN 103232545 B CN103232545 B CN 103232545B
- Authority
- CN
- China
- Prior art keywords
- fusion rotein
- recombinant swine
- interferon
- swine interferon
- recombinant
- 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
- 230000004927 fusion Effects 0.000 title claims abstract description 130
- 230000014509 gene expression Effects 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 31
- 241000282898 Sus scrofa Species 0.000 title claims description 156
- 102000014150 Interferons Human genes 0.000 title claims description 122
- 108010050904 Interferons Proteins 0.000 title claims description 122
- 229940079322 interferon Drugs 0.000 title claims description 122
- 241000588724 Escherichia coli Species 0.000 claims abstract description 37
- 210000003000 inclusion body Anatomy 0.000 claims abstract description 26
- 108010047761 Interferon-alpha Proteins 0.000 claims description 76
- 102000006992 Interferon-alpha Human genes 0.000 claims description 75
- 230000006698 induction Effects 0.000 claims description 34
- 241000894006 Bacteria Species 0.000 claims description 27
- 238000004153 renaturation Methods 0.000 claims description 26
- 238000001556 precipitation Methods 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 20
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 17
- 230000000968 intestinal effect Effects 0.000 claims description 16
- 239000000872 buffer Substances 0.000 claims description 15
- 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 14
- 238000000703 high-speed centrifugation Methods 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 238000004925 denaturation Methods 0.000 claims description 9
- 230000036425 denaturation Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 5
- 239000012139 lysis buffer Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 4
- 238000010612 desalination reaction Methods 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 239000012460 protein solution Substances 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 206010010254 Concussion Diseases 0.000 claims description 2
- 230000009514 concussion Effects 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 230000000392 somatic effect Effects 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 5
- 239000012153 distilled water Substances 0.000 claims 3
- 239000011159 matrix material Substances 0.000 claims 3
- 108010053070 Glutathione Disulfide Proteins 0.000 claims 2
- 230000003115 biocidal effect Effects 0.000 claims 2
- 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 claims 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims 1
- 239000004202 carbamide Substances 0.000 claims 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims 1
- 230000000840 anti-viral effect Effects 0.000 abstract description 8
- 238000001727 in vivo Methods 0.000 abstract description 7
- 230000009466 transformation Effects 0.000 abstract description 7
- 230000006870 function Effects 0.000 abstract description 5
- 102100040019 Interferon alpha-1/13 Human genes 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 4
- 230000001629 suppression Effects 0.000 abstract description 3
- 230000000259 anti-tumor effect Effects 0.000 abstract description 2
- 230000004663 cell proliferation Effects 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 abstract description 2
- 230000002519 immonomodulatory effect Effects 0.000 abstract description 2
- 230000009465 prokaryotic expression Effects 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 abstract 1
- 108020004705 Codon Proteins 0.000 description 39
- 230000009182 swimming Effects 0.000 description 36
- 239000000523 sample Substances 0.000 description 24
- 239000006166 lysate Substances 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 15
- 101710135378 pH 6 antigen Proteins 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 102000037865 fusion proteins Human genes 0.000 description 9
- 108020001507 fusion proteins Proteins 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 241000700605 Viruses Species 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 238000001502 gel electrophoresis Methods 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 5
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 241000711975 Vesicular stomatitis virus Species 0.000 description 5
- 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 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 241000588722 Escherichia Species 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 108060003951 Immunoglobulin Proteins 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
- 239000012930 cell culture fluid Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 210000002706 plastid Anatomy 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000001073 sample cooling Methods 0.000 description 3
- 238000002525 ultrasonication Methods 0.000 description 3
- OJHZNMVJJKMFGX-RNWHKREASA-N (4r,4ar,7ar,12bs)-9-methoxy-3-methyl-1,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;2,3-dihydroxybutanedioic acid Chemical compound OC(=O)C(O)C(O)C(O)=O.O=C([C@@H]1O2)CC[C@H]3[C@]4([H])N(C)CC[C@]13C1=C2C(OC)=CC=C1C4 OJHZNMVJJKMFGX-RNWHKREASA-N 0.000 description 2
- 108091062157 Cis-regulatory element Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 241000057444 Lactobacillus brevis subsp. coagulans Species 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- SPTYHKZRPFATHJ-HYZXJONISA-N dT6 Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)CO)[C@@H](O)C1 SPTYHKZRPFATHJ-HYZXJONISA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000032625 disorder of ear Diseases 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 208000019420 lymphoid neoplasm Diseases 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 108010068617 neonatal Fc receptor Proteins 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Abstract
The invention provides a kind of restructuring Po IFNα 1-Fc fusion rotein and encoding gene, expression, purifying and renaturing inclusion bodies method, belong to biological gene engineering field.IFN α 1 is a modal interferoid, and it has antiviral, antitumor, function such as suppression hematopoietic cell proliferation and immunomodulatory etc. significantly.But natural Po IFNα 1 is very micro-at machine expression in vivo, is difficult to directly a large amount of in body extraction for clinical study and application, and there is removing speed defect faster in blood plasma.For this reason, the invention provides a kind of restructuring Po IFNα 1-Fc fusion rotein being suitable for escherichia coli prokaryotic expression system.Wherein, Po IFNα 1 part is the full sequence of Po IFNα 1 extracellular region, and Fc segment portion comprises the hinge area of antibody, CH2 district and CH3 district, is directly merge between Po IFNα 1 and Fc segment portion.Fusion rotein of the present invention, not only saves most of biological activity of IFN α 1, and extends its transformation period greatly, for its industrialization provides condition.
Description
Technical field
The invention belongs to biotechnology gene field, relate to a kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof, and its expression, purifying and renaturing inclusion bodies method.
Background technology
Interferon, rabbit (Interferon, IFN) is one group of active protein (mainly glycoprotein) with several functions, is a kind of cytokine produced by monocyte and lymphocyte.They have on allogenic cell wide spectrum antiviral, affect Growth of Cells, and the multiple biological activity such as differentiation, immunity moderation function.Different with Induction Condition according to the character of the source of IFN and animal species, cell type, inducer, α, β, γ tri-kinds can be divided into.Wherein IFN-α is that immunocyte is reacted by antiviral response and the close low molecular sugar albumen of the one group of similar produced, function.In numerous hypotype, Interferon α1 is a modal class, it has antiviral, antitumor, function such as suppression hematopoietic cell proliferation and immunomodulatory etc. significantly, is applicable to the treatment of the diseases such as disease of viral infection (as hepatitis), myeloproliferative disease, lymphocytic tumors and other tumour.
China is big country of raising pigs, and pig virus transmissible disease kind is many, harm is large, although the general Pigs Inoculated disease vaccine of current China, but still well can not control epidemic disease.Genetically engineered Recombinant Swine Interferon, rabbit has antiviral effect widely, and have have no side effect, the advantage such as drug residue free, be with a wide range of applications in field of veterinary.Bibliographical information porcine interferon alpha 1 has obvious effect in suppression foot and mouth disease virus vigor, but natural porcine interferon alpha 1 is very micro-at machine expression in vivo, is difficult to directly a large amount of in body extraction for clinical study and application.Therefore the present invention provides a kind of with low cost and can great expression Recombinant Swine Interferon α1 expression system and expression method by genetic engineering means.
On the other hand, as small molecular weight protein, porcine interferon alpha 1 is also the same with other Interferon, rabbit, there is the defect of plasma clearance speed, thus causes in clinical treatment, need repeatedly medication just can reach result for the treatment of.Equally, the frequent of transformation period short and medication also will become the bottleneck using porcine interferon alpha 1 to treat porcine viral diseases.IgG immunoglobulin (Ig) is the main antibody in humans and animals body, and its transformation period is in vivo about 20 days.Its stability is because the Fc fragment of IgG can combine with neonatal Fc receptor (FcRn), avoids IgG to enter in lysosome and is degraded.In view of this, the present invention attempts considering that the Fc fragment increasing IgG on porcine interferon alpha 1 is to form fusion rotein, to improve porcine interferon alpha 1 Half-life in vivo, reaches long-acting object.Thus, the object of the invention is to provide one can retain the original activity of porcine interferon alpha 1, can extend again the porcine interferon alpha 1 of Half-life in vivo and the fusion rotein of Fc fragment.
But, mostly be by the recombinant protein of escherichia coli expression do not dissolve, the intracellular aggregates of non-activity, i.e. inclusion body.The renaturation of inclusion body is a very complicated process, if denaturing conditions is not suitable for there will be the mispairing of intramolecular disulfide bond, intermolecular covalent attachment or hydrophobic binding form polymer, reduce the ratio motility rate of recombinant protein on the one hand, cause quality product defective, produce Precipitation again simultaneously, affect yield.Therefore, another technical problem to be solved by this invention adopts suitable method that the inclusion bodies of protein of escherichia coli expression is carried out renaturation.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, by codon optimized mode, provide a kind of can the recombination fusion protein of high expression and its gene and expression in intestinal bacteria, purifying, refolding method.
The invention provides Recombinant Swine Interferon α1-Fc fusion rotein, described fusion rotein comprises porcine interferon alpha 1 part and Fc segment portion, wherein, porcine interferon alpha 1 part is the full sequence of porcine interferon alpha 1 extracellular region, Fc segment portion comprises hinge area, CH2 district and CH3 district, is directly to merge between porcine interferon alpha 1 and Fc segment portion.
Fc fragment is wherein selected from the immunoglobulin Fc of human or animal, and be Fc total length or partial sequence, Fc is selected from IgG, IgM, IgD, IgA, and often kind of immunoglobulin class comprises each hypotype, as IgG1, IgG2, IgG3, IgG4.Fc segment portion in fusion rotein of the present invention is particularly preferably from the IgG1 of pig.
Preferably, Recombinant Swine Interferon α1-Fc fusion rotein of the present invention has the aminoacid sequence shown in SEQIDNO:2, and wherein 2-167 amino acids residue is the extracellular domain sequence of porcine interferon alpha 1, and 168-397 amino acids residue is pig IgG 1 sequence.
The invention provides the gene of Recombinant Swine Interferon α1-Fc fusion rotein described above of encoding, its base sequence is as shown in SEQIDNO:1.This sequence aims at escherichia expression system to carry out the codon optimized sequence obtained, and can significantly improve the expression of heterologous gene in Host Strains by contrast.
Present invention also offers the plasmid of the gene containing coding Recombinant Swine Interferon α1-Fc fusion rotein described above, described plasmid is preferably prokaryotic expression plasmid, most preferably is pET21b carrier.
Present invention also offers the coli strain including plasmid described above, preferably, described bacterial strain is selected from e. coli bl21 (DE3) bacterial strain.
Present invention also offers Recombinant Swine Interferon α1-Fc fusion rotein in escherichia coli expression method, comprise the steps:
Steps of the method are:
1. picking one or more contain the E. coli clones of Recombinant Swine Interferon α1-Fc fusion rotein described above, access LB nutrient solution, in 37 DEG C of overnight incubation;
2. get appropriate overnight culture, access in the LB nutrient solution of 100 times amount of described overnight culture, be cultured to mid-log phase OD in 37 DEG C of concussions
600=1.0;
3. in culture, add the IPTG that concentration is 1mmol/L, in 37 DEG C, after abduction delivering 4h, in 4 DEG C with rotating speed 5000rpm, centrifugal treating 15min, collect the coli somatic containing Recombinant Swine Interferon α1-Fc fusion rotein.
All containing penbritin 50-100 μ g/mL in described LB nutrient solution.
Expression method described above of the present invention is through the repeated multiple times experiment of contriver and gropes and verify the effective means the most for expressing Recombinant Swine Interferon α1-Fc fusion rotein obtained, the expression amount of the method is high, and express obtain renaturing inclusion bodies after activity higher.
Present invention also offers the inclusion body purification method of Recombinant Swine Interferon α1-Fc fusion rotein, comprise the steps:
1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine Interferon α1-Fc fusion rotein intestinal bacteria, resuspended with the PBS of precooling, and in 4 DEG C of high speed centrifugation process; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer BufferA3-10mL, stirs damping fluid, thalline is hanged.
3. it be the PMSF of 100mmol/L, 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL that every gram (weight in wet base) thalline adds 3-10 μ L concentration, in stirring on ice.
4. broken thalline, sample is placed on ice, ultrasonic, and in 4 DEG C of high speed centrifugation process.
5. precipitation lavation buffer solution BufferB washing, and in 4 DEG C of high speed centrifugation process, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer BufferC dissolves, stirred at ambient temperature 30-60min.
7. the fully rear room temperature high speed centrifugation process of mixing, abandons precipitation, gets supernatant, namely obtain Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln.
This purification process preferred steps is as follows:
1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine Interferon α1-Fc fusion rotein intestinal bacteria, resuspended with the PBS of precooling, in 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer BufferA5mL, stirs, thalline is hanged with slicking glass rod.
3. it is the PMSF of 100mmol/L that every gram (weight in wet base) thalline adds 5 μ L concentration, and 5 μ L concentration are the N,O-Diacetylmuramidase of 100mg/mL, stir 20min on ice.
4. with the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.In 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm, abandon supernatant.
5. precipitation lavation buffer solution BufferB washing, in 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer BufferC dissolves, stirred at ambient temperature 30min.
7. fully after mixing under room temperature with the centrifugal 15min of the rotating speed of 12000rpm, abandon precipitation, get supernatant, namely obtain Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln.
Present invention also offers the renaturing inclusion bodies method of the Recombinant Swine Interferon α1-Fc fusion rotein after optimization, comprise the steps:
Get the Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln that appropriate denaturation buffer BufferC dissolves, with renaturation buffer BufferD, protein concentration is diluted to 0.2mg/mL, when 4 DEG C of renaturation are to 24h, by the 0.45 μm of membrane filtration of recombinant protein solution after renaturation, namely obtain the Recombinant Swine Interferon α1-Fc fusion rotein solution of lower concentration.And can further with the ultrafiltration desalination of molecular weight cut-off 10KDa, concentrated, low-temperature vacuum drying, namely obtains Recombinant Swine Interferon α1-Fc fusion rotein powder.Composition and the content thereof of each damping fluid are as shown in the table:
Present invention also offers Recombinant Swine interferon alpha 1 – Fc fusion rotein treat in preparation and prevent the purposes in the medicine of porcine reproductive and respiratory syndrome, porcine influenza and pig blue-ear disease disease.In porkling treatment of diseases, Recombinant Swine interferon alpha 1 – Fc fusion rotein can nonspecific performance antiviral effect widely, raising immune response and the defence capability strengthened virus.Meanwhile, Recombinant Swine interferon alpha 1 – Fc fusion rotein also can with other vaccine conbined usage, alleviate the untoward reaction of vaccine, strengthen overall antiviral, bacterium, parasitic effect.Recombinant Swine interferon alpha 1 – Fc fusion rotein also can with multiple cause of disease vaccine conbined usage, can immune level be significantly improved, minimizing stress, reduce the side effect of vaccine, thus reach stronger therapeutic action.
Of the present invention through optimised Recombinant Swine Interferon α1-Fc fusion rotein recombination sequence, be more suitable for the expression of escherichia expression system, expressed Recombinant Swine Interferon α1-Fc fusion rotein is far above the expression amount of porcine interferon alpha 1-Fc fusion rotein native gene sequence at escherichia expression system, and, compared with Recombinant Swine Interferon α1, Recombinant Swine Interferon α1-Fc fusion rotein of the present invention on the basis that ensure that Recombinant Swine Interferon α1 activity largely on extend its transformation period in vivo, achieve the long-acting object with avoiding medication repeatedly.Simultaneously, provided by the present invention is a boar source type fusion rotein, be more suitable for the preparation for Swine virus disease veterinary drug, and the procaryotic cell expression of this fusion rotein provided by the present invention, purifying and refolding method also control its preparation cost greatly, for its industrialization provides guarantee.
Accompanying drawing explanation
Fig. 1 represents Recombinant Swine Interferon α1-Fc fusion rotein codon optimized front and back nucleotide sequence comparison
Wherein, odd-numbered line (row that namely " original series " is corresponding) is porcine interferon alpha 1-Fc fusion rotein natural gene nucleotide sequence, i.e. codon optimized front sequence; Even number line (i.e. " majorizing sequence " corresponding row) is the gene nucleotide series of Recombinant Swine Interferon α1-Fc fusion rotein of the present invention, the sequence after namely codon optimized.
Fig. 2-a, Fig. 2-b are the restructuring codon optimized front and back of porcine interferon alpha 1-Fc fusion rotein CAI index in escherichia coli expression host.
Wherein, Fig. 2-a represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized before in escherichia coli expression host CAI index be 0.61; Fig. 2-b represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized after in escherichia coli expression host CAI index be 0.86.
Fig. 3-a, Fig. 3-b are restructuring porcine interferon alpha 1-Fc fusion rotein codon optimal codon frequency distribution areal map in escherichia coli expression host.
Wherein, Fig. 3-a represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized before in escherichia coli expression host optimal codon frequency distribution areal map, as can be seen from the figure: the poor efficiency codon (<30%) of the codon optimized presequence of Recombinant Swine Interferon α1-Fc fusion rotein occurs that per-cent is 10%; Fig. 3-b represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized after in escherichia coli expression host optimal codon frequency distribution areal map, the poor efficiency codon (<30%) of the codon optimized presequence of Recombinant Swine Interferon α1-Fc fusion rotein occurs that per-cent is 0.
Fig. 4-a, Fig. 4-b in restructuring porcine interferon alpha 1-Fc fusion rotein codon in escherichia coli expression host average GC base contents distributed areas figure.
Wherein, Fig. 4-a represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized before in escherichia coli expression host average GC base contents be: 59.32%; Fig. 4-b represent Recombinant Swine Interferon α1-Fc fusion rotein codon optimized after in escherichia coli expression host average GC base contents be: 54.10%.
Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of the codon optimized front and back mRNA of restructuring porcine interferon alpha 1-Fc fusion rotein.
The secondary structure prediction figure of the codon optimized premessenger RNA of Fig. 5-a Recombinant Swine Interferon α1-Fc fusion rotein, Fig. 5-b are the secondary structure prediction figure of the codon optimized rear mRNA of restructuring porcine interferon alpha 1-Fc fusion rotein.
Fig. 6 is restructuring porcine interferon alpha 1-Fc fusion protein expression plasmid building process figure.
Fig. 7 is restructuring porcine interferon alpha 1-Fc fusion rotein optimized gene agarose gel electrophoresis figure.
Wherein, swimming lane 1 is for containing the Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene PCR primer of NdeI and XhoI restriction enzyme site in two ends; Swimming lane 2 is 500bpDNALadder; Swimming lane 3 is the pET21b carrier after NdeI and XhoI double digestion.
Fig. 8 is the SDS-PAGE gel electrophoresis figure of restructuring porcine interferon alpha 1-Fc fusion rotein and corresponding western blot figure.
Fig. 8-a is restructuring porcine interferon alpha 1-Fc fusion protein S DS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is the Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate not adding IPTG induction; Swimming lane 3 is for adding the Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate of IPTG induction.
Fig. 8-b is restructuring porcine interferon alpha 1-Fc fusion protein immunization trace figure.
Wherein, swimming lane 1(10-230KDa) the albumen loading Marker of pre-dyed of wide region, swimming lane 2 is the Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate not adding IPTG induction: swimming lane 3 is for adding the Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate of IPTG induction.
Fig. 9 Recombinant Swine Interferon α1-Fc fusion rotein high expression optimum induction SDS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is 0.5mmol/LIPTG induction 1h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 3 is 0.5mmol/LIPTG induction 2h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 4 is 0.5mmol/LIPTG induction 3h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 5 is 0.5mmol/LIPTG induction 4h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 6 is 1mmol/LIPTG induction 1h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 7 is 1mmol/LIPTG induction 2h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 8 is 1mmol/LIPTG induction 3h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 9 is 1mmol/LIPTG induction 4h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 10 is 1.5mmol/LIPTG induction 1h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 11 is 1.5mmol/LIPTG induction 2h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 12 is 1.5mmol/LIPTG induction 3h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate; Swimming lane 13 is 1.5mmol/LIPTG induction 4h Recombinant Swine Interferon α1-Fc fusion rotein E. coli lysate.
Figure 10 is the porcine interferon alpha 1-Fc fusion rotein inclusion body SDS-PAGE electrophorogram after renaturation
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 contains the full bacterium lysate of Recombinant Swine Interferon α1-Fc fusion rotein for ultrasonic degradation; Swimming lane 3 is by Recombinant Swine Interferon α1-Fc fusion rotein inclusion body precipitation after BufferB first time cleaning; Swimming lane 4 is Recombinant Swine Interferon α1-Fc fusion rotein inclusion body precipitation after the cleaning of BufferB second time; Swimming lane 5 is Recombinant Swine Interferon α1-Fc fusion rotein supernatant solution after B renaturation.
Figure 11 is restructuring Interferon α1-Fc stability western blot figure in porcine blood serum.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is not for add porcine interferon alpha 1 and recombinantinterferonα 1-Fc fusion rotein pig anteserum sample; Swimming lane 3 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 0h; Swimming lane 4 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 1h; Swimming lane 5 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 2h; Swimming lane 6 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 8h; Swimming lane 7 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 24h; Swimming lane 8 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 72h; Swimming lane 9 is for adding the pig anteserum sample of 1 μm of ol/mL porcine interferon alpha 1 and 1 μm of ol/mL Recombinant Swine Interferon α1-Fc fusion rotein 120h.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further, should be understood that quoting embodiment is only not used in for illustration of the present invention and limits the scope of the invention.
embodiment 1 Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene optimization design
Contriver is according to the published porcine interferon alpha 1(Susscrofainterferon of GenBank, alpha1) cDNA sequence (GenBank accession number: NM_214393.1) and the middle hinge area of the cDNA sequence (GenBank accession number: NM_213828.1) of pig IgG Fc fragment (SusscrofaIgGheavychain), CH2 district and CH3 district, these 2 genes are directly merged and carries out the codon optimized gene obtaining Recombinant Swine Interferon α1-Fc fusion rotein of the present invention, as shown in SEQIDNo:1.
Here is carried out codon optimized to restructuring porcine interferon alpha 1-Fc fusion rotein, and before and after optimizing, each parameter comparison is as follows:
1. codon adaptation indexI (CAI)
From Fig. 2-a, before codon is not optimized, Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene codon adaptation indexI (codonadaptationindex, CAI) in intestinal bacteria is 0.61.From Fig. 2-b, after codon optimized, Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene CAI index in intestinal bacteria is made to be 0.86.Be considered to this gene during usual CAI=1 is optimal high expression state in this expression system, CAI index is lower shows that this gene expression level in this host is poorer, therefore can find out have passed through codon optimized after the gene order that obtains can improve the expression level of Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene in intestinal bacteria.
2. optimal codon frequency of utilization (FOP)
From Fig. 3-a, based on coli expression carrier, before codon is not optimized, the poor efficiency codon of porcine interferon alpha 1-Fc antigen-4 fusion protein gene sequence occurs that per-cent is 10%.The gene that this is not optimized adopts series connection rare codon, and these codons may reduce translation efficiency, even can dismiss translation assemblage.From Fig. 3-b, after codon optimized, Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene occurs that in E. coli system the frequency of poor efficiency codon is 0.
3.GC base contents (GCcurve)
GC content ideal distribution region is 30%-70%, all can affect to some extent transcribe and translation efficiency at this any peak of extra-regional appearance.Contrasted from the GC base average content distributed areas figure of the porcine interferon alpha 1-Fc antigen-4 fusion protein gene of Fig. 4-a, Fig. 4-b, be 59.32% by showing in porcine interferon alpha 1-Fc antigen-4 fusion protein gene GC base average content before optimization in Fig. 4-a, by demonstrating the GC of the sequence elimination after optimization content in Fig. 4-b in extra-regional 60 bases of 30%-70%, after being finally optimized, the GC base average content of Recombinant Swine Interferon α1-Fc fusion rotein is 54.10%.
3. cis-acting elements
| Cis-acting elements | Before optimization | After optimization |
| E.coli_RBS(AGGAGG) | 4 | 0 |
| PolyT(TTTTTT) | 0 | 0 |
| PolyA(AAAAAAA) | 0 | 0 |
| Ch site (GCTGGTGG) | 0 | 0 |
| T7Cis(ATCTGTT) | 0 | 0 |
4. remove tumor-necrosis factor glycoproteins
The secondary structure prediction figure of 5.mRNA
After DNA is transcribed into mRNA, because mRNA is strand linear molecule, by folded back on itself, complementary base pair is met, by the hairpin structure (Hairpin) of hydrogen bonded.5 ' hairpin structure can play regulating and controlling effect in the translation initiation stage.If but hairpin structure is very long, the required energy that unwinds is very high, just likely has influence on translation.So need the sequence expressed should avoid long and that energy is high hairpin structure as far as possible.After codon optimized, from the secondary structure prediction figure of Fig. 5-a, the codon optimized front and back mRNA of Fig. 5-b porcine interferon alpha 1-Fc fusion rotein, 5 ' hairpin structure after optimization and the required energy that unwinds are more suitable for the expression of target protein.
embodiment 2: the expression plasmid of Recombinant Swine Interferon α1-Fc antigen-4 fusion protein gene builds
By the fragment that Recombinant Swine Interferon α1-Fc fusion rotein full genome (as shown in SEQIDNo:1) after optimizing synthesizes, be building up in pUC57 plasmid (Jin Sirui Science and Technology Ltd. provides by Nanjing), obtain one and preserve plasmid for a long time, be designated as pUC57-pIFN α 1-Fc plasmid.With pUC57-pIFN α 1-Fc plasmid for template, upstream and downstream introduce NdeI and XhoI restriction enzyme site respectively, carry out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1:GGAATTCCATATGTGTGACCTGCCGCAAACG
Downstream primer:
P2:CCGCTCGAGTCATTTGCCCTGGGTTTTGGAG
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and archaeal dna polymerase PhusionHigh-FidelityDNApolymerase(used is purchased from Theromo-Fisherscientific), 2U/ μ L, adds 0.5 μ L.Reaction conditions is 98 DEG C of 5s, 55 DEG C of 45s, 72 DEG C of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and product size is consistent with expection size (1194bp).(as shown in Figure 7)
The gene product DNA gel obtained is reclaimed test kit (purchased from Beijing Tian Gen biochemical technology company limited) purifying.After purifying, with NdeI and XhoI(purchased from NewEnglandBiolabs company) double digestion, with T4 ligase enzyme (purchased from NewEnglandBiolabs company), the product after double digestion is connected in pET21b plasmid (purchased from Merck company), be transformed in DH5 α competent cell (purchased from Beijing Tian Gen biochemical technology company limited), 37 DEG C of overnight incubation in the LB flat board of the penbritin (purchased from Amresco company) containing 100 μ g/mL.Second day screening positive clone bacterium, order-checking, comparison result display is completely the same with expected sequence, namely obtains the expression plasmid of a kind of form of Recombinant Swine Interferon α1-Fc fusion rotein, is designated as pET21b-pIFN α 1-Fc.
the high expression of embodiment 3 Recombinant Swine Interferon α1-Fc fusion rotein in intestinal bacteria and qualification
Concrete steps are as follows:
1. by pET21b-pIFN α 1-Fc Plastid transformation correct for comparison of checking order in embodiment 2 in e. coli bl21 (DE3) competence bacterial strain (purchased from Beijing Tian Gen biochemical technology company limited), incubated overnight in 37 DEG C of ampicillin plate.
2. within second day, choose 1-4 the restructuring bacterium colony containing pET21b-pIFN α 1-Fc plasmid, the LB nutrient solution (purchased from Amresco company) of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.
3. get overnight culture in 50 μ L steps 2, access 5mL contains the LB nutrient solution of 100 μ g/mL penbritins, 37 DEG C of shaking culture.
4. survey bacterium liquid OD every 1h after inoculation
600value, treats OD
600when=1.0, with the IPTG(of 1mmol/L purchased from Amresco company) carry out abduction delivering.
5. collect bacterium liquid after abduction delivering 4h, high speed centrifugation (rotating speed: 12000rpm) 3min, by the PBS washing and precipitating of precooling, adds 5 × sds gel sample loading buffer, and 100 DEG C of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm) 1min, gets supernatant.Do not add the recombination bacillus coli culture of IPTG by this step process yet.
6. respectively get 10 μ L do not add IPTG and add IPTG induction by step 5 process after sample, 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis, moves to bottom separation gel to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining and immunoblotting, observes expression product band, sees Fig. 8-a and Fig. 8-b.
embodiment 4 Recombinant Swine Interferon α1-Fc fusion rotein high expression optimum induction
Much research shows that cell growth rate has a strong impact on the expression of foreign protein, therefore must to inoculation amount of bacteria, culture temperature, after cell growth time and induction, cell density control before induction, overgrowth or overrun and all can affect the expression amount of Recombinant Swine Interferon α1-Fc fusion rotein inclusion body in intestinal bacteria.Use Three factors four level, set up IPTG concentration and induction time orthogonal table, by SDS-PAGE gel electrophoresis analysis induction Recombinant Swine Interferon α1-Fc fusion protein expression.
Concrete steps are as follows:
1. by pET21b-pIFN α 1-Fc Plastid transformation correct for comparison of checking order in embodiment 2 to BL21(DE3) in competence bacterial strain (purchased from Beijing Tian Gen biochemical technology company limited), incubated overnight in 37 DEG C of ampicillin plate.
2. next day, picking 1-4 the restructuring bacterium colony containing pET21b-pIFN α 1-Fc plasmid, the LB nutrient solution of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.
3. get overnight culture access 5mL in 50 μ l steps 2 and contain the LB induction broth of 100 μ g/mL penbritins, 37 DEG C of shaking culture.
4. survey bacterium liquid OD after inoculation
600value, treats OD
600when=1.0, adding concentration according to following table is that 0.5mmol/L, 1.0mmol/L, 1.5mmol/LIPTG carry out abduction delivering.
Table 1 investigates inducer concentrations and the induction time of expression of recombinant proteins
5.1,2,3, collect Recombinant Swine Interferon α1-Fc fusion rotein bacterium liquid after 4h successively, high speed centrifugation (rotating speed: 12000rpm) 3min, by the PBS washing and precipitating of precooling, add 5 × sds gel sample loading buffer, 100 DEG C of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm) 1min.
6. get the Recombinant Swine Interferon α1-Fc fusion rotein culture suspension that 10 μ L do not add IPTG induction and add different concns IPTG, different induction time, 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis, moves to bottom separation gel to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining, observes Recombinant Swine Interferon α1-Fc fusion protein expression products band.(see figure 9)
9. the expression of Recombinant Swine Interferon α1-Fc fusion rotein content qualification porcine interferon alpha 1-Fc fusion rotein is expressed in the analysis of gel imaging system thin layer scanning.Finally determine that the applicable inductive condition that this is implemented is 1mmol/LIPTG, induction time is 4h.
embodiment 5 Recombinant Swine Interferon α1-Fc fusion rotein inclusion body purification and renaturation
1. will collect the intestinal bacteria precipitation containing induction Recombinant Swine Interferon α1-Fc fusion rotein obtained in embodiment 3, resuspended with the PBS of precooling, in 4 DEG C with 12000rpm, centrifugal 15min; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer BufferA5mL, stirs, thalline is hanged with slicking glass rod.
3. every gram (weight in wet base) thalline adds 5 μ L100mmol/LPMSF, and 5 μ L100mg/mL N,O-Diacetylmuramidases, stir 20min on ice.
4. with the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.4 DEG C, 12000rpm, centrifugal 15min.
5. precipitation lavation buffer solution BufferB washing, 4 DEG C, 12000rpm, centrifugal 15min, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer BufferC dissolves, stirred at ambient temperature 30min.
7. room temperature 12000rpm after fully mixing, centrifugal 15min, abandons precipitation, gets supernatant, namely obtains Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln.
8. adopt dilution refolding method to carry out renaturation to the Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln in step 7.
Dilution refolding: get the Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln that appropriate denaturation buffer BufferC dissolves, with bio-rad company of the QuickStartBradford1xDyeReagent(U.S.) survey its concentration, then with renaturation buffer BufferD, protein concentration is diluted to 0.2mg/mL, when 4 DEG C of renaturation are to 24h, the 0.45 μm of filter membrane (MerckMillipore company) of recombinant protein solution after renaturation is filtered, namely obtains the Recombinant Swine Interferon α1-Fc fusion rotein solution of lower concentration.With super filter tube (MerckMillipore company) desalination of molecular weight cut-off 10KDa, concentrated, in vacuum freeze drier (Beijing Sihuan Scientific Instrument Factory Co., Ltd) low-temperature vacuum drying, namely obtain Recombinant Swine Interferon α1-Fc fusion rotein powder.
Each damping fluid according to the form below preparation:
The each buffer components of table 2
9. carry out SDS-PAGE gel electrophoresis (Figure 10) with lavation buffer solution BufferB twice washed product in step 5 respectively, at the visible obviously band of object scope.
the preparation of embodiment 6 Recombinant Swine Interferon α1(in detail can see the earlier application of applicant: 201310025176.8)
Concrete steps are as follows:
1. build the recombination bacillus coli can expressing Recombinant Swine Interferon α1.
According to the published porcine interferon alpha 1(Susscrofainterferon of GenBank, alpha1) cDNA sequence (GenBank accession number: NM_214393.1), according to escherichia expression system to this gene carry out codon optimized after obtain Recombinant Swine Interferon α1 gene, as shown in SEQIDNo:3.By the fragment that the Recombinant Swine Interferon α1 full genome after optimizing synthesizes, be building up in pUC57 plasmid, obtain pUC57-prIFN α 1 plasmid.
With pUC57-prIFN α 1 plasmid for template, upstream and downstream primer introduces NdeI and XhoI restriction enzyme site respectively, carries out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1：GGAATTCCATATGTGTGACCTGCCGCAAACGC
Downstream primer:
P2：CCGCTCGAGTCATTCCTTTTTGCGCAGACGATC
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and archaeal dna polymerase PhusionHigh-FidelityDNApolymerase, 2U/ μ L used, adds 0.5 μ L.Reaction conditions is 98 DEG C of 5s, 55 DEG C of 20s, 72 DEG C of 30s, 25 circulations.PCR primer with NdeI and XhoI double digestion, is connected in pET21b plasmid with T4 ligase enzyme, and is increased in DH5 α competent cell by Plastid transformation after reclaiming kits with DNA gel.The expression plasmid pET21b-prIFN α 1 of amplification is transformed in escherichia coli BL21(DE3) expression bacterial strain.
2. the expression of Recombinant Swine Interferon α1 inclusion body
By e. coli bl21 (DE3) incubated overnight in 37 DEG C of ampicillin plate containing pET21b-prIFN α 1.Choose 1-4 the restructuring bacterium colony containing pET21b-prIFN α 1 plasmid next day, the LB nutrient solution of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.Get the LB induction broth of 50 μ L overnight culture access 5mL containing 100 μ g/mL penbritins, 37 DEG C of shaking culture.Treat OD
600when=1.0, induce with the IPTG of 1mmol/L.Bacterium liquid is collected, high speed centrifugation, by the PBS washing and precipitating of precooling after 4h.
3. the renaturation of Recombinant Swine Interferon α1 inclusion body and purifying
By resuspended for the PBS of the PBS washing and precipitating precooling through precooling in step 2, in 4 DEG C with 12000rpm, centrifugal 15min; Repeat once.Abandon supernatant, add lysis buffer BufferA5mL by every gram (thalline weight in wet base), thalline is hanged.Every gram of (thalline weight in wet base) thalline adds 5 μ L100mmol/LPMSF, and 5 μ L100mg/mL N,O-Diacetylmuramidases, stir 20min on ice.With the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.4 DEG C, 12000rpm, centrifugal 15min.Precipitation lavation buffer solution BufferB washing, 4 DEG C, 12000rpm, centrifugal 15min, precipitation inclusion body, repeats once.Inclusion body precipitation denaturation buffer BufferC dissolves, stirred at ambient temperature 30min.Room temperature 12000rpm after abundant mixing, centrifugal 15min, abandons precipitation, gets supernatant, namely obtains Recombinant Swine Interferon α1 denaturing soln.Adopt dialysis renaturation method renaturation inclusion body: use denaturation buffer BufferC by Recombinant Swine Interferon α1 denaturing soln concentration dilution to 0.2mg/mL, inject the dialysis card of molecular weight cut-off 10KDa, 4 DEG C of dialysis renaturations, change a renaturation buffer BufferD every 6h.When renaturation is to 24h, recombinant protein solution after renaturation is crossed 0.45 μm of filter membrane, namely obtain the Recombinant Swine Interferon α1 renaturation solution of lower concentration.With the super filter tube desalination of molecular weight cut-off 10KDa, concentrated, in vacuum freeze drier low-temperature vacuum drying, namely obtain Recombinant Swine Interferon α1 powder.Each buffer is as shown in table 2.Recombinant Swine Interferon α1 powder 4 DEG C of refrigerators are deposited stand-by.
embodiment 7 Recombinant Swine Interferon α1-Fc fusion rotein Determination of biological activity
(VSV, its TCID50 are 5 × 10 to pig vesicular stomatitis virus
7/ 100 μ L, academy of agricultural sciences of Guangdong Province veterinary institute provides) can infected pigs nephrocyte (PK-15, academy of agricultural sciences of Guangdong Province veterinary institute provides), the present invention utilizes the Antiviral Mechanism of porcine interferon alpha 1 to detect at the defense reaction of porcine kidney cell to pig vesicular stomatitis virus under porcine interferon alpha 1 or Recombinant Swine Interferon α1-Fc fusion rotein existence condition, porcine interferon alpha 1 or Recombinant Swine Interferon α1-Fc fusion rotein is obtained to the protective effect curve of PK-15 cell by the pathology situation detecting PK-15 cell, thus measure Recombinant Swine Interferon α1 or Recombinant Swine Interferon α1-Fc fusion rotein biologic activity.
1). prepared by positive reference substance solution: (pig genetically engineered recombinant cytokine: IFN-LLS-2, purchased from Hong Kong Man Pu animal nutrition company limited to get pig interferon positive reference substance.), after by specification redissolves, with the MEM cell culture fluid (Gibico product) containing 6% foetal calf serum by 10 times of one-level stepwise dilutions.
2). prepared by Recombinant Swine Interferon α1-Fc fusion protein sample solution: to take in embodiment 5 Recombinant Swine Interferon α1-Fc fusion rotein dry powder sample 500ug after renaturation, do 10 times of one-level stepwise dilutions with cell culture fluid, obtain 50 μ g/mL, 5 μ g/mL, 0.5 μ g/mL, 5 × 10 respectively
-2μ g/mL, 5 × 10
-3μ g/mL, 5 × 10
-4μ g/mL recombinantinterferonα 1 – Fc fusion rotein solution.
4). on 96 orifice plates, every hole adds positive controls sample or the Recombinant Swine Interferon α1-Fc fusion protein sample solution of different concns, then (cell concn is about 1.8 × 10 to add the fresh PK-15 cell suspension gone down to posterity of 50 μ L
6-2.2 × 10
6individual/mL), total system 100 μ L, 37 DEG C, 5%CO
2hatch about 24h, PK-15 cell attachment under condition and grow to individual layer.Discard cell culture supernatant, every hole adds the MEM nutrient solution of 100 μ L containing 2% foetal calf serum of the VSV virus of 100 TCID50.Set up negative control virus group (only add the virus of PK-15 cell and same dose, do not add restructuring porcine interferon alpha 1) and blanc cell control group (only add PK-15 cell and cell culture fluid, do not add restructuring porcine interferon alpha 1 and virus) simultaneously.37 DEG C, 5%CO
224h is cultivated, observations when the cytopathy 90%-100% of virus control wells under condition.Be a unit (being designated as " U ") to cause the amount of the cytopathic Interferon, rabbit in half hole.
Experimental result shows, and obvious pathology all occurs the cell in negative control virus group, and in blanc cell control group, Growth of Cells is normal.Compared with positive control, when the concentration of Recombinant Swine Interferon α1-Fc fusion rotein is at 1 μ g/mL-0.1ug/mL, starts to produce and eliminate the phenomenon of VSV virus on the impact of PK-15 cell normal growth.Converted by result and determine that tiring of positive control Interferon, rabbit is 3.2 × 10
4it is 1.2 × 10 that U/mL, Recombinant Swine Interferon α1-Fc fusion protein sample is tired
3u/mL.As can be seen here, Recombinant Swine Interferon α1-Fc of the present invention has antiviral activity, show wide application prospect in porkling disease prevention and treatment use reverse side, and preparation technology is simple, makes may being achieved of suitability for industrialized production long-acting genetically engineered Recombinant Swine Interferon α1-Fc.
the stability test of embodiment 8 Recombinant Swine Interferon α1-Fc fusion rotein
In order to detect the stability of porcine interferon alpha 1-Fc fusion rotein in serum, contriver is specially designed a kind of in-vitro simulated porcine blood serum environment, and the embodiment 5 Recombinant Swine of the present invention Interferon α1-Fc fusion rotein preparing Recombinant Swine Interferon α1 and 1 μm of ol/ml in the embodiment 6 of 1 μm of ol/ml is placed in fresh porcine blood serum 50 μ L, 37 DEG C jointly, 120rpm, react 0 respectively, 1,2,8,-20 DEG C are preserved after 24,72,120h.With mouse-anti Recombinant Swine Interferon α1 monoclonal antibody (IFNalphaAntibody (K9), CatalogNumber:271001, ThermoPierce) for primary antibodie carries out protein immunoblot test to the Recombinant Swine Interferon α1 in porcine blood serum and Recombinant Swine Interferon α1-Fc fusion rotein, as shown in Figure 11 porcine interferon alpha 1 after reaction 24h by the enzymic digestion in serum totally, and Recombinant Swine Interferon α1-Fc fusion rotein 120h in serum still keeps stable.As can be seen here, porcine interferon alpha 1-Fc fusion rotein prepared by the present invention has higher stability compared with Recombinant Swine Interferon α1, extends its Half-life in vivo, reaches the effect of long-acting administration.
Claims (8)
1. encode the gene of Recombinant Swine Interferon α1-Fc fusion rotein, its base sequence is as shown in SEQIDNO:1.
2. a carrier, described carrier contains the gene of claim 1.
3. carrier as claimed in claim 2, described carrier is pET21b.
4. intestinal bacteria, described intestinal bacteria have the carrier of Claims 2 or 3.
5. intestinal bacteria as claimed in claim 4, described intestinal bacteria are BL21(DE3) bacterial strain.
6. a procaryotic cell expression method for Recombinant Swine Interferon α1-Fc fusion rotein, comprises the steps:
(1) in substratum, the intestinal bacteria described in claim 4 or 5 are cultivated under suitable conditions;
(2) from intestinal bacteria and/or substratum, be separated restructuring porcine interferon alpha 1-Fc fusion rotein.
7. expression method as claimed in claim 6, comprises the steps:
(1) picking is one or more containing the E. coli clones described in claim 4 or 5, and access contains antibiotic LB nutrient solution, overnight incubation;
(2) get overnight culture to transfer in containing in antibiotic fresh LB nutrient solution, be cultured to mid-log phase OD in 37 DEG C of concussions
600=1.0;
(3) in culture, add the IPTG that concentration is 1mmol/L, 37 DEG C, after abduction delivering 4h, the coli somatic that centrifugal treating is collected containing Recombinant Swine Interferon α1-Fc fusion rotein precipitates.
8. the purifying of Recombinant Swine Interferon α1-Fc fusion rotein and a refolding method, is characterized in that, comprise following steps:
(1) precipitate collecting obtain as claimed in claim 7 containing induction Recombinant Swine Interferon α1-Fc fusion rotein intestinal bacteria, PBS is resuspended, and in 4 DEG C of high speed centrifugation process, repeats once;
(2) suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer BufferA3-10mL, stirs damping fluid, thalline is hanged;
(3) it be the PMSF of 100mmol/L, 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL that every gram (weight in wet base) thalline adds 3-10 μ L concentration, in stirring on ice;
(4) broken thalline, sample is placed on ice, ultrasonic, and in 4 DEG C of high speed centrifugation process;
(5) precipitation lavation buffer solution BufferB washing, and in 4 DEG C of high speed centrifugation process, precipitation inclusion body, repeats once;
(6) inclusion body precipitation denaturation buffer BufferC dissolves, stirred at ambient temperature 30-60min;
(7) the fully rear room temperature high speed centrifugation process of mixing, abandons precipitation, gets supernatant, namely obtain Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln;
(8) the Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln that appropriate denaturation buffer BufferC dissolves is got, with renaturation buffer BufferD by the concentration dilution of Recombinant Swine Interferon α1-Fc fusion rotein denaturing soln to 0.2mg/mL, 4 DEG C of dialysis renaturation 24h, by the 0.45 μm of membrane filtration of recombinant protein solution after renaturation, namely obtain Recombinant Swine Interferon α1-Fc fusion rotein renaturation solution;
The further ultrafiltration and concentration of described Recombinant Swine Interferon α1-Fc fusion rotein renaturation solution, desalination are to minimum volume, and low-temperature vacuum drying, namely obtains Recombinant Swine Interferon α1-Fc powder;
The composition of described BufferA is 50mmol/LTris – HCl, 0.2mol/LNaCl, 1mmol/LEDTA and 0.1mmol/LPMSF, and solution matrix is distilled water, and pH is 7.5;
The composition of described BufferB is 50mmol/LTris – HCl, 0.2mol/LNaCl, 1mmol/LEDTA, 0.5%(v/v) TritonX-100 and 0.5mol/L urea, solution matrix is distilled water, and pH is 7.5;
The composition of described BufferC is 50mmol/LTris – HCl, 0.2mol/LNaCl, 1mmol/LEDTA, 0.1mmol/LPMSF, 7mol/L Guanidinium hydrochloride and 20mmol/LDTT, and solution matrix is distilled water, and pH is 7.8;
The composition of described BufferD is 50mmol/LTris – HCl, 1mmol/LEDTA, GSH and GSSG, the mol ratio 8:1 of GSH and GSSG.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310182855.6A CN103232545B (en) | 2013-05-16 | 2013-05-16 | A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310182855.6A CN103232545B (en) | 2013-05-16 | 2013-05-16 | A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103232545A CN103232545A (en) | 2013-08-07 |
| CN103232545B true CN103232545B (en) | 2016-03-16 |
Family
ID=48880621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310182855.6A Active CN103232545B (en) | 2013-05-16 | 2013-05-16 | A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103232545B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103570836B (en) * | 2013-10-25 | 2016-03-16 | 江苏众红生物工程创药研究院有限公司 | A kind of Recombinant Swine interferon beta 1-Fc fusion rotein and encoding gene thereof and expression method |
| CN107082814A (en) * | 2017-06-12 | 2017-08-22 | 扬州大学 | A kind of long-acting interferon and preparation method thereof |
| CN107254482A (en) * | 2017-07-18 | 2017-10-17 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving Recombinant Swine interferon alpha fusion protein antiviral activity |
| CN107326037A (en) * | 2017-07-18 | 2017-11-07 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving Recombinant Swine interferon beta fusion protein antiviral activity |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967196A (en) * | 2010-11-10 | 2011-02-09 | 夏志南 | Interferon fusion protein, preparation thereof and application thereof |
| CN103059122A (en) * | 2013-01-23 | 2013-04-24 | 江苏众红生物工程创药研究院有限公司 | Recombined porcine interferon alpha 1, as well as gene encoding gene and expression method thereof |
| CN103087200A (en) * | 2013-01-28 | 2013-05-08 | 江苏众红生物工程创药研究院有限公司 | Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein |
-
2013
- 2013-05-16 CN CN201310182855.6A patent/CN103232545B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967196A (en) * | 2010-11-10 | 2011-02-09 | 夏志南 | Interferon fusion protein, preparation thereof and application thereof |
| CN103059122A (en) * | 2013-01-23 | 2013-04-24 | 江苏众红生物工程创药研究院有限公司 | Recombined porcine interferon alpha 1, as well as gene encoding gene and expression method thereof |
| CN103087200A (en) * | 2013-01-28 | 2013-05-08 | 江苏众红生物工程创药研究院有限公司 | Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103232545A (en) | 2013-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103570836B (en) | A kind of Recombinant Swine interferon beta 1-Fc fusion rotein and encoding gene thereof and expression method | |
| CN103087200B (en) | Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein | |
| CN103059124B (en) | Recombined porcine interferon Gamma, coding gene thereof and expressing method | |
| CN103232545B (en) | A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method | |
| CN102146135A (en) | Recombinant human-like collagen and production method thereof | |
| CN102041263B (en) | Chicken alpha interferon/interleukin 2 chimeric gene | |
| CN103193887B (en) | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein | |
| CN103319608B (en) | Pig IFNalpha1-Fc fusion protein as well as encoding gene and expression method thereof | |
| CN103265637B (en) | A kind of Recombinant Swine IL-4-Fc fusion rotein and encoding gene thereof and expression method | |
| CN103757041B (en) | A kind of methods and applications preparing pig source Interferon, rabbit-λ 3 | |
| CN103059122B (en) | Recombined porcine interferon alpha 1, as well as gene encoding gene and expression method thereof | |
| CN103232544B (en) | Recombination porcine interferon gamma-Fc fusion protein as well as coding gene and expression method thereof | |
| CN103012577B (en) | Recombinant porcine interleukin 4, and encoding gene and expression method thereof | |
| CN106399266A (en) | Recombinant baculovirus for expressing dog serum albumin fused interferon gamma and application of recombinant baculovirus | |
| CN103012578B (en) | Recombinant porcine interleukin 2, and encoding gene and expression method thereof | |
| CN109627316A (en) | Recombinant protein and the application of 2 gene of grass carp IFN-γ and its coding | |
| CN103059123B (en) | Recombinant porcine interferon beta1, encoding gene and expression method thereof | |
| CN102492714A (en) | Method for preparing recombinant Chinese rural dog interferon-alpha | |
| CN101570757A (en) | Porcine alpha interferon and interleukin 2 chimeric gene, construction method and protein purification method thereof | |
| CN101544693B (en) | Recombined extrasin alpha 1 two-strand body protein and preparation method thereof | |
| CN108948163A (en) | Queensland nut plant alexin and its application | |
| CN101434958A (en) | Method for preparing porcine alpha-interferon | |
| CN103014015B (en) | Gene segment for coding porcine interferon-gamma and application of gene segment | |
| CN105755008A (en) | DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon | |
| CN117304344B (en) | Fusion protein, recombinant strain expressing fusion protein and application of recombinant strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP02 | Change in the address of a patent holder |
Address after: 213125 Jiangsu city of Changzhou Province Cloud River New District No. 518 Changzhou Qianhong biochemical pharmaceutical biological all red Patentee after: ZonHon Biopharma Institute Inc. Patentee after: Changzhou Gensun Institute of Biomedicine Co., Ltd. Address before: 213022 the Yellow River Middle Road, Xinbei District, Jiangsu, China, No. 132, No. Patentee before: ZonHon Biopharma Institute Inc. Patentee before: Changzhou Gensun Institute of Biomedicine Co., Ltd. |
|
| CP02 | Change in the address of a patent holder |
Address after: 213125, Yunhe Road, Xinbei District, Jiangsu, Changzhou, 518 Co-patentee after: Changzhou Gensun Institute of Biomedicine Co., Ltd. Patentee after: ZonHon Biopharma Institute Inc. Address before: 213125 Jiangsu city of Changzhou Province Cloud River New District No. 518 Changzhou Qianhong biochemical pharmaceutical biological all red Co-patentee before: Changzhou Gensun Institute of Biomedicine Co., Ltd. Patentee before: ZonHon Biopharma Institute Inc. |
|
| CP02 | Change in the address of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 213125 No. 518 Yunhe Road, Xinbei District, Changzhou City, Jiangsu Province Co-patentee after: Jiangsu Jingsen Biomedical New Materials Technology Co., Ltd. Patentee after: ZonHon Biopharma Institute Inc. Address before: 213125 No. 518 Yunhe Road, Xinbei District, Changzhou City, Jiangsu Province Co-patentee before: Changzhou Gensun Institute of Biomedicine Co., Ltd. Patentee before: ZonHon Biopharma Institute Inc. |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190514 Address after: 213125 No. 518 Yunhe Road, Xinbei District, Changzhou City, Jiangsu Province Patentee after: Jiangsu Jingsen Biomedical New Materials Technology Co., Ltd. Address before: 213125 No. 518 Yunhe Road, Xinbei District, Changzhou City, Jiangsu Province Co-patentee before: Jiangsu Jingsen Biomedical New Materials Technology Co., Ltd. Patentee before: ZonHon Biopharma Institute Inc. |
|
| TR01 | Transfer of patent right |