Method for preparing swine-original interferon-gamma 3 and application
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A pig-derived, gene-based technology, applied in the field of genetic engineering product preparation, can solve the problems of lagging research on type III IFN and achieve high PEDV activity
Active Publication Date: 2014-04-30
GUANGDONG MAIKETE BIOLOGICAL TECH
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[0003] At present, scholars at home and abroad have conducted in-depth research on the biological functions a
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Embodiment 1
[0031] Embodiment 1, the preparation method of porcine source IFN-λ3, carries out the following steps successively:
[0032] (1) Construct the cloning vector:
[0033] ① Induced by poly(I:C) (1.5ug, 2h) cultured ST cells (1×10 5 / ml), the induced cultured cells were harvested, and the total RNA of the cells was extracted with Trizol reagent;
[0034] ② Design specific primers based on the only porcine IFN-λ3 gene sequence (GQ996936) published on GenBank, and design specific primers (S1 and S2) for amplifying the full-length porcine IFN-λ3 gene,
[0035] Upstream primer S1: 5'-ATGGCCCTGGGTGGCTCG-3'
[0036] Downstream primer S2: 5'-TCAGACACACAGGTCTCC-3';
[0037] ③Use RT-PCR technology to amplify the full length of porcine IFN-λ3 gene (use PrimeScript RT-PCR Kit for PCR amplification); RT-PCR reaction conditions are 50°C for 30min; 94°C for 3min; 94°C for 30s, 55°C 30s, 40s at 72°C, 35 cycles; 10min at 72°C;
[0038] ④ By transforming Top10 Escherichia coli, the porcine IF...
Embodiment 2
[0065] Example 2, the activity assay of the pig-derived IFN-λ3 prepared in Example 1 of the present invention:
[0066] Using the microcytopathic inhibition method, MDBK cells (MDBK bovine kidney cells) / VSV (vesicular stomatitis virus) are used as the basic detection system. One interferon unit (U) was defined as the highest dilution of interferon that inhibited 50% cytopathic effect (CPE). The results of the activity test showed that the biological activity of pig-derived IFN-λ3 was 2×10 3 U / mg. At present, all expressed type I and type II interferons are detected by MDBK cells / VSV to detect the expression activity of interferon, so we also use this system to analyze whether the IFN-λ3 we express has biological activity, and then analyze the anti-virus activity Activity
Embodiment 3
[0067] Embodiment 3, anti-PEDV virus (porcine epidemic diarrhea virus) effect of porcine source IFN-λ3 protein
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Abstract
The invention discloses a method for preparing swine-original interferon-gamma 3 (IFN-gamma 3) protein and application, belonging to the field of preparation of a genetic engineered product. The method comprises the steps of: cloning a swine-original interferon-gamma 3 coding gene, building a prokaryotic expression vector which contains the swine-original interferon-gamma 3 gene having no signal peptide sequence, and transforming escherichia coli and carrying out induced expression by using the recombined prokaryotic expression vector containing the swine-original interferon-gamma 3 gene, wherein the optimal induction temperature is 20 degrees centigrade, the optimal induction time is 16 hours, and the optimal IPTG (Isopropyl-beta-d-Thiogalactoside) concentration is 0.4mM. A product prepared by the method disclosed by the invention provides basis for researching the biological activity and the action mechanism of the swine-original interferon-gamma 3, and also can be applied to preparation of swine porcine epidemic diarrhea virus prevention drugs.
Description
technical field [0001] The invention belongs to the field of preparation of genetic engineering products, and in particular relates to a method and application for preparing porcine interferon-λ3 (IFN-λ3) protein. Background technique [0002] Interferon (IFN) is a highly biologically active glycoprotein secreted by recipient cells after human and animal cells are infected by viruses, or affected by nucleic acids, bacterial endotoxins, and mitogens. Interferon has the advantages of biological activity, quick-acting multipotency, and non-toxicity, and has become an ideal biological product for the prevention and treatment of swine viral diseases. In addition to its antiviral activity, interferon also has immunoregulatory effects, and can be used as an immune adjuvant to improve the immune activity of vaccines. According to the biological activity and antigenic activity of interferon, it can be divided into three types: IFN-α, IFN-β, IFN-γ, and IFN-α and IFN-β with common sur...
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