CN1544629A - Method for preparing recombinant goose interleukin-2 protein and its application - Google Patents
Method for preparing recombinant goose interleukin-2 protein and its application Download PDFInfo
- Publication number
- CN1544629A CN1544629A CNA2003101086228A CN200310108622A CN1544629A CN 1544629 A CN1544629 A CN 1544629A CN A2003101086228 A CNA2003101086228 A CN A2003101086228A CN 200310108622 A CN200310108622 A CN 200310108622A CN 1544629 A CN1544629 A CN 1544629A
- Authority
- CN
- China
- Prior art keywords
- gsil
- goose
- expression
- interleukin
- product
- 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.)
- Granted
Links
- 108010002350 Interleukin-2 Proteins 0.000 title claims abstract description 46
- 241000272814 Anser sp. Species 0.000 title claims abstract description 39
- 102000000588 Interleukin-2 Human genes 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title abstract description 13
- 239000000047 product Substances 0.000 claims abstract description 45
- 230000014509 gene expression Effects 0.000 claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 35
- 230000008521 reorganization Effects 0.000 claims description 28
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 12
- 210000004698 lymphocyte Anatomy 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 9
- 238000001742 protein purification Methods 0.000 claims description 9
- 230000000968 intestinal effect Effects 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 7
- 238000013467 fragmentation Methods 0.000 claims description 7
- 238000006062 fragmentation reaction Methods 0.000 claims description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000000568 immunological adjuvant Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 4
- 230000036039 immunity Effects 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- RCQMOSJJIVCPJO-UHFFFAOYSA-N 2-(5-methyl-2,4-dioxopyrimidin-1-yl)ethoxymethylphosphonic acid Chemical compound CC1=CN(CCOCP(O)(O)=O)C(=O)NC1=O RCQMOSJJIVCPJO-UHFFFAOYSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000011146 sterile filtration Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims 2
- 230000003248 secreting effect Effects 0.000 claims 2
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 claims 1
- 230000000840 anti-viral effect Effects 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 230000005611 electricity Effects 0.000 claims 1
- 230000000590 parasiticidal effect Effects 0.000 claims 1
- 239000002297 parasiticide Substances 0.000 claims 1
- 239000012264 purified product Substances 0.000 claims 1
- 230000000392 somatic effect Effects 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 6
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 4
- 239000012043 crude product Substances 0.000 abstract 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract 1
- 239000000969 carrier Substances 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 abstract 1
- 238000005336 cracking Methods 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000002245 particle Substances 0.000 abstract 1
- 241000287828 Gallus gallus Species 0.000 description 18
- 230000004071 biological effect Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 235000019750 Crude protein Nutrition 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 206010035148 Plague Diseases 0.000 description 5
- 241000607479 Yersinia pestis Species 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 241001452677 Ogataea methanolica Species 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 230000003393 splenic effect Effects 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 230000000527 lymphocytic effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 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 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000009795 derivation Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000272808 Anser Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000224483 Coccidia Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- -1 gan Species 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 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 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000003526 lymphopoietic effect Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003257 protein preparation method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant goose interleukin-2(gsIL-2) protein preparing method and its use. The DNA fraction of RT-PCR amplified Zhedong white goose interleukin-2, prokaryotic expression carrier pBAD/HisB, and eukaryotic expression carriers pMET alpha B and pMETB compose expression particles pBAD/HisB/gsIL-2, pMET alpha B/gsIL-2 and pMETA/gsIL-2, which are converted to Escherichia coli LMG194, microzyme PMAD11 and PMAD16, respectively, then inducing expression. After inducing, making ultrasonic wave cracking and deposit elimination by centrifugation on Escherichia coli and microzyme PMAD16 to obtain crude products of gsIL-2 protein, PMAD11 expressed gsIL-2 is mainly separated in the culture medium, centrifugating and taking supernatant to obtain gsIL-2 protein crude products. Using Ni-NTAArgarrose protein purifying system, thus able to faster obtain gsIL-2 pure product, and the crude and pure products can act as immune assistant and disease-curing drugs. It applied genetic engineering technique to prepare recombinant protein gsIL-2, and has simple technical flow, low production cost, good stability, high bioactivity and other characters.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the proteic preparation method and its usage of a kind of reorganization goose interleukin II.
Background technology
1.Schauenstein found at first that chicken splenic lymphocyte secretion is a kind of and had an active cytokine of the mammalian interleukin of being similar to element-2 sample Deng (1982), subsequently, the using sulfated ammonium precipitation of Schnetlzler etc. (1983), the bicarbonate of ammonia resuspension, Sephadex G-100 gel-filtration obtains two classes and has active isolate, and its molecular weight lays respectively between 9～11.5KDa and the 19.5～21.5Kda.After this Vainio etc. use and obtained molecular weight with quadrat method is the molecule of 13KDa, and they estimate that with isoelectrofocusing the PI of chicken IL-2 is 5.9.Frederickson etc. (1987) use Phenyl-sepharose, anionresin and superose-12 and cross methods such as post, isolate two classes and have active material, the one molecular weight is 14KDa, its two molecular weight is about 26～30KDa, the latter is stronger than the former activity, and both all have low wetting ability.Myers (1992) reported a kind of from the chicken splenic lymphocyte nutrient solution that ConA stimulates " five-step approach " of purifying chicken IL-2, i.e. (1) gel-filtration tentatively concentrates the chicken splenic lymphocyte suspension that ConA stimulates; (2) ammonium sulfate precipitation; (3) with Sephadex G-100 chromatography; (4) (high performanceliquid chromatography is HPLC) with (5) Phenyl-sepharose chromatography for the high back voltage liquid chromatography (LC).Purified IL-2 albumen carries out the SDS-PAGE electrophoresis under reductive condition, the albumen that obtains has two peak values, and molecular weight is about 36～39KDa and 17.5～25KDa, with the albumen alkanisation reduction on peak, use Sephadex G-100 chromatography again, can produce low-molecular-weight peak.
Sundick etc. (1997) obtain chicken IL-2 gene first by the method in construction cDNA library from chicken spleen, Choi etc. (1998) are cloned into prokaryotic expression carrier PUC18 and carrier for expression of eukaryon pcDNA3 to the chicken IL-2 gene that contains long 430 Nucleotide of signal sequence respectively, express in intestinal bacteria and CHO-K1 cell respectively, two kinds of albumen that obtain all have the lymphopoietic biologic activity of the chicken T of promotion.Stepaniak etc. (1999) compare deep research to chicken IL-2 vivoexpression, the chicken IL-2 gene of removing leading peptide is expressed in intestinal bacteria Pgex-2t system, the albumen that obtains has the proteic biologic activity of natural chicken IL-2, but expression efficiency is low only to be 63 μ g/L, and mostly be non-solubility albumen, be unfavorable for proteic recovery and purifying.Be head it off, they have adopted pET32a
+Efficient expression system is brought up to 4mg/L with expressing quantity, and the overwhelming majority is a soluble proteins.Simultaneously, this scholar complete encoder block that will contain leading chicken IL-2 gene is cloned into carrier for expression of eukaryon pCR3.1
+In, in the kidney fibroblast, express, found that this system not only can stably express chicken IL-2 albumen, and go up cleer and peaceful purified albumen and all have good biologic activity, thereby proved that chicken IL-2 is similar with Mammals IL-2, its activity does not also rely on leader sequence.
2. chicken IL-2 precursor protein is made up of 143 amino acid, and sophisticated albumen is made up of 121 amino acid.It and Mammals IL-2 have similar biologic activity, as promoting bone-marrow-derived lymphocyte and the lymphocytic propagation of T, strengthen the functions such as killing activity of NK cell, play a significant role in immunity of organism.Research based on chicken IL-2, we utilize the increased dna fragmentation of East Zhejiang province white goose IL-2 of RT-PCR technology from splenic lymphocyte, and realized that efficiently expressing in protokaryon and eukaryotic cell, expression product have the biologic activity of stimulation goose spleen lymphocyte proliferation.
Summary of the invention
The purpose of this invention is to provide the proteic preparation method and its usage of a kind of reorganization goose interleukin II.
The step of reorganization goose interleukin II protein preparation method is as follows:
1) dna fragmentation that in the white goose monokaryon lymphocyte of East Zhejiang province, clones goose interleukin II (gsIL-2) with contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/HisB/gsIL-2; Perhaps above-mentioned gsIL-2DNA fragment with contain P
ADGIThe carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2; Perhaps above-mentioned gsIL-2 dna fragmentation with contain P
ADGIThe carrier for expression of eukaryon pMETB of promotor is set up into type expression vector pMET B/gsIL-2 in the born of the same parents;
2) use RM as basic medium, by fermenting and amplifying colibacillus engineering LMG194;
3) use BMDY as basic medium,, add nutrient substances such as nitrogenous source, carbon source at the expression different time by fermenting and amplifying Yeast engineering bacteria PMAD11 and PMAD16;
4) intestinal bacteria LMG194 and yeast PMAD16 induces after ultrasonic treatment, the centrifugal precipitation of going are the gsIL-2 raw product, the gsIL-2 that PMAD11 expresses mainly secretes in substratum, is the gsIL-2 raw product through the centrifuging and taking supernatant.Can obtain the pure product of gsIL-2 with Ni-NTA Argarose protein purification system;
5) after raw product or pure product add stablizer such as glycine and solubility promoter, make finished product after sterile filtration, packing and the freeze-drying, be stored in-20 ℃ the cold storage environment.
Reorganization gsIL-2 albumen is used for enhance immunity function and immunological adjuvant; Be used for the treatment of the palmipeds disease;
Advantage of the present invention: the gsIL-2 albumen of (1) escherichia coli expression mainly exists in the genetic engineering bacterium with the form of fusion rotein, through ultrasonic wave target protein is discharged, do not need other processing just can obtain the gsIL-2 crude protein, and protein stability is good, biologic activity is higher; (2) select secretor type Yeast expression carrier pMET α B for use, expressed gsIL-2 albumen major part is secreted in the substratum, and not only without any need for processing just can obtain the gsIL-2 raw product, and target protein has satisfactory stability and biologic activity; (3) select nonsecreting type Yeast expression carrier pMET B for use, the gsIL-2 albumen of expression mainly exists in the engineering bacteria with the form of fusion rotein, just can obtain the gsIL-2 crude protein through ultrasonication, and crude protein has than advantages of higher stability and biologic activity; (4) used Ni-NTA Agarose protein purification system is simple to operate, the purity of protein height that obtains.(5) reorganization gsIL-2 is remarkable as immunological adjuvant and disease therapeuticing medicine effect.
Embodiment
1 goose interleukin-2 gene clone stimulates goose spleen monocyte with canavailn (ConA), the Trizol single stage method is extracted total RNA, by RT-PCR method amplification with cloned East Zhejiang province white goose interleukin II cDNA fragment, be connected to sequencing vector pGEM-T Easy Vector, measure the gsIL-2 dna sequence dna.
The pcr amplification product subclone of the colibacillary structure of 2 recombinant expressed goose inactivated interleukin-2 genetic engineerings goose interleukin II is in a new expression vector pBAD/His B, be built into gsIL-2 prokaryotic expression carrier pBAD/His B/gsIL-2, it can be expressed in intestinal bacteria LMG194, make gsIL-2 albumen account for 9～10% of bacterial protein, expression rate does not reduce through going down to posterity more than 100 times.
The saccharomycetic structure of 3 recombinant expressed goose inactivated interleukin-2 genetic engineerings with the pcr amplification product subclone of goose interleukin II in novel carrier for expression of eukaryon pMET α B and pMET B, be built into gsIL-2 expression vector pMET α B/gsIL-2 and pMET B/gsIL-2 respectively, with BMDY as basic medium, with methyl alcohol as inductor, by optimizing dissolved oxygen, concussion speed, processing parameters such as fermentation pH value, can make the gsIL-2 gene obtain expressing in P.methanolica expression strain PMAD11 and PMAD16, expression amount accounts for 20～25% and 10～12% of bacterial protein respectively., expression rate does not reduce through going down to posterity more than 100 times.
The preparation and the purifying process of 4 reorganization goose interleukin II albumen raw product.
The preparation of the raw product of being set up and purifying process flow process are simple, good stability, active high.This technology has following characteristics: the gsIL-2 albumen of (1) escherichia coli expression mainly exists in the engineering bacteria with the form of fusion rotein, through ultrasonic wave target protein is discharged, do not need other processing just can obtain the gsIL-2 crude protein, and its biologic activity is higher; (2) select secretor type Yeast expression carrier pMET α B for use, expressed gsIL-2 albumen major part is secreted in the substratum, and not only without any need for processing just can obtain the thick product of gsIL-2 albumen, and target protein has good biologic activity; (3) select nonsecreting type Yeast expression carrier PMET B for use, the gsIL-2 albumen of expression mainly exists in the engineering bacteria with the form of fusion rotein, just can obtain the gsIL-2 crude protein through ultrasonication, and crude protein has than higher biologic activity; (4) used Ni-NTA Agarose protein purification system is simple to operate, the purity of protein height that obtains.
5 raw product or pure product add solubility promoters such as phosphoric acid buffer, with the degerming of microvoid membrane filtration, can be used as immunological adjuvant and disease therapeuticing medicine.
The important biomolecule of IL-2 is learned function and is promoted the lymphocytic propagation of T exactly, we use the goose spleen T lymphocyte that stimulates through ConA as target cell, with mtt assay the proteic activity of reorganization gsIL-2 is detected, confirm that expression product has higher biologic activity.
The present invention uses recombinant DNA technology, adopts the reverse transcription PCR method to obtain a kind of reorganization gsIL-2 albumen.Through downstream purification, the product under the pilot scale has reached the specification of quality of clinical application.
The design of embodiment 1. Oligonucleolide primers is with synthetic
According to the duck of registering among the GenBank (AF294323), chicken (AF017645) and turkey (AJ007463) IL-2 nucleotide sequence, design 1 pair of RT-PCR primer:
Upstream primer, 5 '-AATACTAGCACAGAGACAACCAG-3 '
Downstream primer, 5 '-TTACTGAAATTTATTAAATATCATCTA-3 '
The lymphocytic separation of embodiment 2. spleen monokaryons
1 monthly age of aseptic collection East Zhejiang province white goose spleen, shred and place no Ca
2+, Mg
2+(717mmol/L K among the ionic PBS
2HPO
4, 283mmol/L KH
2PO
4PH7.2), 4 ℃ of centrifugal 10min of 300 * g, get 4 ℃ of centrifugal 30min of supernatant 500 * g and collect lymphocyte, wash 2 times with PBS then, with RPMI1640 nutrient solution (not containing calf serum) washing 1 time, behind trypan blue (0.1%) the dyeing viable count, cell is made into 1 * 10 again with RPMI1640 grown cultures liquid (containing 10% calf serum, the penicillin of 100IU/ml and the Streptomycin sulphate of 100 μ g/ml)
7The cell suspension of/ml.Add 10mg/L final concentration ConA in the cell suspension, the packing Tissue Culture Plate, 5% CO2gas incubator is cultivated 16h for 40 ℃ and is collected lymphocyte culture Trizol reagent single stage method extraction cell total rna.
Embodiment 3.RT-PCR amplifying target genes fragment
Goose IL-2 cDNA first chain is synthesized in First Strand cDNA Synthesis Kit reverse transcription; PCR is carried out in transcription product 30 circulations under 95 ℃ of 1min, 47 ℃ of 45s, 72 ℃ of 2min environment, and last 72 ℃ are extended 10min.The PCR product is through 1% agarose gel electrophoresis analysis.
The structure and the sequencing of embodiment 4. sequencing vectors
Reclaim purpose PCR product fragment with PCR Purification test kit purifying, utilize T-A clone strategy to be directly connected in pGEM-T Easy carrier, connect product Transformed E .coli TOP10 competent cell, at the dull and stereotyped enterprising row filter of the LB that contains penbritin, X-gal and IPTG.PCR and enzyme are cut the evaluation positive colony.Select 5 positive colonies to check order.East Zhejiang province white goose interleukin-2 gene Nucleotide and derivation aminoacid sequence see Table 1.
1 AATACTAGCACAGAGACAACCAGAACACTGACAAGATGTGCAAAGTACTGATCTTCAGCTGCCTTTCGG 69
2 M C K V L I F S C L S 69
1 TAGTAATGCTTATGACTACAGCTTATGGAGCACCTCTATCAGAGAAAAATGACACTCTTACAACTTTAA 138
2 V V M L M T T A Y G A P L S E K N D T L T T L 138
1 TAAAAGATTTAGAAAAGCTGGGAACAAGCATGAAGAAGATTAATCTTGAGCTCTACACACCAAATGAGA 207
2 I K D L E K L G T S M K K I N L E L Y T P N N 207
1 AACAGGAGTGCAGCTGGCAAACTCTACAATGTTACTTGAAAGAAATAGTCACCTTGGAGAATGAAATTG 276
2 K Q E C S W Q T L Q C Y L K E I V T L N E N I 276
1 AAGATGAGGATGAAATTGAAGATGAGAACGTATTTAGTGTTAGGAATATTGAAAAGAATCTCCAAAAAC 345
2 E D E D E I E D E N V F S V R N I E K N L Q K 345
1 TTACGGACCTAATTCCCCCAGGAACGGGATGCAAGATCTGTGAAGCTAATGACAAGAAGGAGTTTCCTG 414
2 L T D L I P P G T G C K I C E A N D K K E F P 414
1 AATTTCACCGAGAGCTGACCAACTTTCTGAGATCTATGCTAAAATAAGCAGATGACCATTTATATTTTT 483
2 E F H R E L T N F L R S M L K - 483
1 ACTGCTACGTTATTTATTTAAGTATTTAATTACAAATAATTTATATATTTTACCCTGGGGCTAACTAAC 552
1 TTGCTGTCCATTTTGGGACCACTGTATGCTCTTAGTGTGGGTGATAACGTGTCTGTTCTAAGATCACAT 621
1 TTGATCCTTTCTGTAAGCCCTACGGGCTCAAAATGTATGTTGGAAAACTAATTGGTTCTCACTTTGTCA 690
1 GTAAACTTAAAATGACAACTATTTATTGAAAGAATTGTTAAAAGTTACTTGTAGATGATATTTAATAAA 759
1 TTTCAGTAA 768
Table 1. East Zhejiang province white goose interleukin-2 gene Nucleotide and derivation aminoacid sequence (SEQ No.1)
Embodiment 5.pBAD/His B/gsIL-2 expression vector establishment, screening and evaluation
According to East Zhejiang province white goose interleukin II sequence of having cloned, synthetic upstream and downstream primer: the UP1 (5 ' GC at maturation protein sequence two ends
GAATTCGCACCTCTATCAGAGAA3 '), 5 ' end contains the EcoRI restriction enzyme site; DOWN1 (5 ' GC
AAGCTTTTATTTTAGCATAGATCT 3 '), 5 ' end contains the HandIII restriction enzyme site; Being template with pGEM-T-Easy/gsIL-2 under 95 ℃ of 1min, 47 ℃ of 45s, 72 ℃ of 2min environment, PCR is carried out in 30 circulations, extends 10min at last.After PCR product electrophoresis reclaims, behind EcoRI, HandIII double digestion, recovery purpose fragment is also cloned in the EcoRI+HandIII enzyme of expression plasmid pBAD/His B and is cut in the window, make up recombinant expression plasmid pBAD/His B/gsIL-2, transform host bacterium TOP10, extract recombinant plasmid and carry out PCR, double digestion evaluation and sequencing.
The fermentative production (intestinal bacteria) of embodiment 6. reorganization gsIL-2
Recombinant plasmid through identifying changes LMG194 host bacterium over to, the picking mono-clonal is inoculated in the RM substratum that contains penbritin and glucose, 37 ℃ of overnight incubation, in 1: 100 ratio culture is inoculated in the RM substratum, the 250rpm concussion was cultivated about 2 hours, make OD600 ≈ 0.5, added the L (+) of 20% concentration-pectinose inducing culture 3 hours in 1: 100 ratio, centrifugal collection thalline.Thalline is added an amount of 1 * SDS sample-loading buffer, adopt 15% SDS-PAGE to identify, the Bradford method is measured the product expression amount can reach 9～10%.
Embodiment 7.pMET α B/gsIL-2 and pMETB/gsIL-2 expression vector establishment, screening and evaluation
According to East Zhejiang province white goose interleukin II sequence, at the synthetic upstream and downstream primer in its maturation protein sequence two ends: upstream primer with example 5 in UP1; Downstream primer DOWN2 (5 ' GC
GGATCCTATTTTAGCATAGATCT3 '), 5 ' end contains the BamHI restriction enzyme site.With pGEM-T-Easy/gsIL-2 is that template is carried out PCR, and amplification condition is with example 5.After PCR product electrophoresis reclaims, behind EcoRI, BamHI double digestion, recovery purpose fragment is also cloned respectively in the EcoRI+BamHI enzyme in expression plasmid pMET α B and pMETB strong promoter downstream and is cut in the window, make up recombinant expression plasmid, transform host bacterium TOP10, extract recombinant plasmid and carry out PCR, double digestion evaluation and sequencing.
Reported method such as employing Choi prepare PMAD11 and PMAD16 competent cell, get 100 μ lPMAD11 and PMAD16 yeast competent cell and 2 μ g respectively and cut completely through the KpnI enzyme that pMET α B/gsIL-2 and pMETB/gsIL-2 mix, use Bio-Rad Gene Pluser at 375V/cm, 25 μ F, transform under the condition of 250 Ω, the YPAD that adds the 1ml room temperature then, hatch 1h for 28～30 ℃, the centrifugal 3min of 1500 * g room temperature collects thalline, again be suspended from 100 μ l, 1 * YNB, bacterium liquid is coated MD to be selected on the flat board, cultivated 3～4 days for 28～30 ℃, observe the growing state of transformant.White colony on the random screening MD flat board extracts pastoris genomic dna with Genomic DNA mini-prepkit and carries out the pcr amplification evaluation.
The embodiment 8. reorganization proteic fermentative production of gsIL-2 (yeast)
Picking PCR is accredited as male PMAD11 and PMAD16 clone and is inoculated in the 20mL BMDY substratum respectively, and 28～30 ℃, 16～18h to OD is cultivated in the 250r/min concussion
600=2～10.Room temperature, the centrifugal 5min of 1500 * g collects thalline and is resuspended in the 10mL BMMY substratum.28～30 ℃, 250r/min continues concussion and cultivated 4 days, adding 5% methyl alcohol in per 24 hours, to make the methyl alcohol final concentration be 0.5%, the centrifugal collection respectively gone up cleer and peaceful precipitation, the SDS-PAGE electrophoresis carries out gsIL-2 and expresses evaluation, it is 20～25% that the Bradford method is measured the expression amount of gsIL-2 in PMAD11, and the expression amount in PMAD16 is 10～12%.
The preservation of embodiment 9. recombination engineering colon bacillus and stability
Engineering bacteria adds 30% glycerine ,-70 ℃ of preservations.Short-term is preserved bacterial classification can use the LB flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out sequencing.The three is identical as a result.Simultaneously 3 generation bacterial classification amplification cultivation thing expression level suitable, prove that the engineering bacteria among the present invention is very stable.
Saccharomycetic preservation of embodiment 10. recombination engineerings and stability
Engineering bacteria adds 30% glycerine ,-70 ℃ of preservations.Short-term is preserved bacterial classification can use the YPAD flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out sequencing.The three is identical as a result.Simultaneously 3 generation bacterial classification amplification cultivation thing expression level suitable, prove that the engineering bacteria among the present invention is very stable.
The preparation and the separation and purification of embodiment 11. reorganization gsIL-2 crude protein
The gsIL-2 albumen of expressing through intestinal bacteria LMG194 mainly exists with the form of fusion rotein, thalline is behind ultrasonic disruption, can obtain gsIL-2 albumen raw product, further the Ni-NTAAgarose protein purification system with QIAGEN company can obtain the pure product of gsIL-2 albumen faster.
The gsIL-2 albumen that contains the P.methanolica expression strain PMAD11 expression of pMET α B/gsIL-2 recombinant vectors is mainly secreted in substratum, can obtain gsIL-2 albumen raw product through the centrifuging and taking supernatant, the gsIL-2 albumen that contains the P.methanolica expression strain PMAD16 expression of pMETB/gsIL-2 recombinant vectors mainly exists in the yeast cell, gsIL-2 albumen raw product can be obtained through ultrasonic treatment, the centrifugal precipitation of going, the pure product of gsIL-2 albumen can be obtained faster with the Ni-NTA Agarose protein purification system of QIAGEN company
The proteic evaluation of embodiment 12. reorganization gsIL-2
The HPLC purity checking is equal more than 98%, mtt assay is measured the biologic activity of raw product and pure product, intestinal bacteria and P.methanolica zymic expression product not only have the activity that stimulates the T lymphocyte growth, and active activity apparently higher than ConA inductive T lymphocyte supernatant liquor.
Embodiment 13. reorganization gsIL-2 albumen physico-chemical property and stability
Reorganization gsIL-2 albumen is more stable to heat, and 56 ℃ 1 hour, 37 ℃ 12 hours or 70 ℃ were handled still retentive activity in 15 minutes, can preserve more than 1 year for 4 ℃, and its activity keeps stablizing in pH2～9 scopes.Reorganization gsIL-2 albumen is to the proteolytic enzyme sensitivity, and is insensitive to DNA enzyme, RNA enzyme.
Embodiment 14. reorganization gsIL-2 albumen are as immunological adjuvant and disease therapeuticing medicine.
Can make goose salmonella typhi organ intrusion rate reduce by 49%～58% with reorganization gsIL-2 albumen, in addition, inject the goose embryo, can significantly reduce the Salmonellas organ intrusion rate of incubation period goose embryo with reorganization gsIL-2 albumen.Aspect parasite,, behind reorganization gsIL-2 albumen subcutaneous injection goose in 1 age in week, can obviously reduce the output of egg capsule behind (p＜0.05) coccidium infection in the day before yesterday or metainfective the 1st day that goose infects coccidia.After 30 age in days geese infect gosling plague; the Effective Vate of Protection of intramuscular injection anti gosling plague serum is 78.2%; the protection ratio that intramuscular injection reorganization gsIL-2 albumen adds anti gosling plague serum is up to 91.5%, and separately intramuscular injection anti gosling plague serum and gosling plague vaccine protection ratio be 64.8% and 2.7%.
Claims (7)
1. reorganization goose interleukin II proteic preparation method is characterized in that its step is as follows:
1) dna fragmentation that in the white goose monokaryon lymphocyte of East Zhejiang province, clones goose interleukin II (gsIL-2) with contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/HisB/gsIL-2; Perhaps above-mentioned gsIL-2DNA fragment with contain P
AUG1The carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2; Perhaps above-mentioned gsIL-2 dna fragmentation with contain P
AUG1The carrier for expression of eukaryon pMETB of promotor is set up into type expression vector pMET B/gsIL-2 in the born of the same parents;
2) use RM as basic medium, by fermenting and amplifying colibacillus engineering LMG194;
3) use BMDY as basic medium,, add nutrient substances such as nitrogenous source, carbon source at the expression different time by fermenting and amplifying Yeast engineering bacteria PMAD11 and PMAD16;
4) intestinal bacteria LMG194 and yeast PMAD16 induces after ultrasonic treatment, the centrifugal precipitation of going are the gsIL-2 raw product, the gsIL-2 that PMAD11 expresses mainly secretes in substratum, is the gsIL-2 raw product through the centrifuging and taking supernatant.Can obtain the pure product of gsIL-2 with Ni-NTA Argarose protein purification system;
5) after raw product or pure product add stablizer such as glycine and solubility promoter, make finished product after sterile filtration, packing and the freeze-drying, be stored in-20 ℃ the cold storage environment.
2. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1,, it is characterized in that said gsIL-2 dna fragmentation and contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/His B/gsIL-2, through electric transformed into escherichia coli LMG194 is the gsIL-2 gene engineering colibacillus, behind L (+)-pectinose abduction delivering, product exists in the engineering bacteria with the form of fusion rotein.
3. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1 is characterized in that above-mentioned said gsIL-2 dna fragmentation and contains P
AUG1The carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2, and PMAD11 is the gsIL-2 gene engineering microzyme through electric shock transformed yeast bacterium, and methanol induction is expressed, and product is present in the engineering bacteria substratum with the secretory product form; PMET α B/gsIL-2 electricity transformed yeast bacterium PMAD16, methanol induction is expressed, and product mainly is present in the engineering bacteria.
4. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying colibacillus engineering LMG194, with RM as basic medium, be by broken coli somatic, with Ni-NTA Argarose protein purification system purified product.
5. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying Yeast engineering bacteria PMAD16, with BMDY as basic medium, expressing nutrient substances such as different time adding nitrogenous source, carbon source, be by broken Yeast engineering bacteria PMAD16 thalline, carry out purifying with Ni-NTA Argarose protein purification system.
6. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying Yeast engineering bacteria PMAD11, with BMDY as basic medium, expressing nutrient substances such as different time adding nitrogenous source, carbon source, Yeast engineering bacteria PMAD11 secretory product carries out purifying with the Ni-NTAArgarose protein purification system.
7. the proteic purposes of reorganization goose interleukin II is characterized in that it is used to improve vaccine immunity as immunological adjuvant and renders a service; As the aquatic bird medicine have antiviral, antibiotic and parasiticide active.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101086228A CN100384998C (en) | 2003-11-11 | 2003-11-11 | Method for preparing recombinant goose interleukin-2 protein and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101086228A CN100384998C (en) | 2003-11-11 | 2003-11-11 | Method for preparing recombinant goose interleukin-2 protein and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1544629A true CN1544629A (en) | 2004-11-10 |
| CN100384998C CN100384998C (en) | 2008-04-30 |
Family
ID=34334779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101086228A Expired - Fee Related CN100384998C (en) | 2003-11-11 | 2003-11-11 | Method for preparing recombinant goose interleukin-2 protein and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100384998C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8632995B2 (en) | 2006-07-27 | 2014-01-21 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN104178494A (en) * | 2013-05-23 | 2014-12-03 | 上海华新生物高技术有限公司 | Preparation process and application of human interleukin 2 (IL-2) |
| CN109091671A (en) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | Goose specificity oligodeoxynucleotide immunologic adjuvant and its application |
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
-
2003
- 2003-11-11 CN CNB2003101086228A patent/CN100384998C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8632995B2 (en) | 2006-07-27 | 2014-01-21 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| US9279000B2 (en) | 2006-07-27 | 2016-03-08 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN105463045A (en) * | 2006-07-27 | 2016-04-06 | 惠氏公司 | High-cell density fed-batch fermentation methods for producing recombinant protein |
| US10301663B2 (en) | 2006-07-27 | 2019-05-28 | Wyeth, Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN104178494A (en) * | 2013-05-23 | 2014-12-03 | 上海华新生物高技术有限公司 | Preparation process and application of human interleukin 2 (IL-2) |
| CN109091671A (en) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | Goose specificity oligodeoxynucleotide immunologic adjuvant and its application |
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
| CN114369152B (en) * | 2022-01-24 | 2023-11-21 | 扬州大学 | Preparation and application of recombinant chicken interleukin-9 protein and antibody thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100384998C (en) | 2008-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1195860C (en) | Delivery of biologically active polypeptides | |
| CN102796200B (en) | Hybrid peptide bursin adjuvant and preparation method and application thereof | |
| CN101870733B (en) | Poultry IL-2 and newcastle disease virus HN gene recombination fusion protein and application thereof | |
| CN115772228A (en) | Duck interferon fusion protein displayed on yeast surface and application thereof | |
| CN1544629A (en) | Method for preparing recombinant goose interleukin-2 protein and its application | |
| CN103012577B (en) | Recombinant porcine interleukin 4, and encoding gene and expression method thereof | |
| CN102492714A (en) | Method for preparing recombinant Chinese rural dog interferon-alpha | |
| CN1778920A (en) | Antibiotic peptide gene and its yeast expression carrier | |
| CN109897857B (en) | Optimized human interleukin-2 gene and its expression method and application | |
| CN1611604A (en) | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method | |
| CN101544693B (en) | Recombined extrasin alpha 1 two-strand body protein and preparation method thereof | |
| CN102180959A (en) | Improved chook Interleukin-2 protein and preparation method thereof | |
| CN1231585C (en) | Method for preparing recombinant duck interleukin-2 protein and its application | |
| CN103012578A (en) | Recombinant porcine interleukin 2, and encoding gene and expression method thereof | |
| CN114540363A (en) | Construction and protein rapid purification method of human-like collagen recombinant pichia pastoris engineering bacteria | |
| CN104418945A (en) | Preparation method of peptide and application of peptide in preparation of medicine and feed additive | |
| CN1224704C (en) | Preparing method for recombinant chBJSTWO protein and use thereof | |
| CN103937828A (en) | Preparation method of fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1 | |
| CN101538572B (en) | Recombinant lactic acid bacteria containing recombinant alpha-2b interferon and application thereof | |
| CN1818068A (en) | Construction of recombinant human leucocyte medium-2(125ser) expression carrier | |
| CN1408863A (en) | Human interleukin-10 gene sequenc and E coli containing the said gene sequence | |
| CN1487083A (en) | Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof | |
| CN100500844C (en) | High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor | |
| CN1258747A (en) | Preparation of 4-tandem thymosin Alpha-1 with colon bacillus | |
| CN1057564C (en) | Microbe capable of producing active recombinant phycobiliprotein and its preparing method |
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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080430 Termination date: 20101111 |