A method for separation and purification of recombinant porcine circovirus capsid protein inclusion body
A technology of porcine circovirus and capsid protein, which is applied to the preparation methods of peptides, chemical instruments and methods, and methods based on microorganisms, etc., can solve the problems such as the lack of thorough research on industrial production, and achieve the effect of avoiding cumbersome steps.
Inactive Publication Date: 2016-05-04
FUZHOU UNIV +1
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In addition, there are currently many reports on porcine circovirus capsid protein as a subunit vaccine, but the research on industrial production is not very thorough
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[0035] 1 Induced expression of recombinant bacteria
[0036] The virus strain used in this laboratory comes from Fuzhou Dabeinong Biotechnology Co., Ltd., which was obtained by amplification.
[0037] The 576bp of the open reading frame ORF2 gene of porcine circovirus type Ⅱ virus, which is integrated into the gene Nco and Xho Two restriction sites, double digestion of the gene, and the same digestion system to digest the pET28a vector, enzyme-linked ORF2 gene and pET28a vector, transformed into the cloned strain TOP10, sequenced and verified, and then introduced the plasmid into the host In Escherichia coli BL-21(DE3), IPTG induces expression, and the expression of the target protein is detected by SDS-PAGE and Western blot analysis, and the target protein exists in the form of inclusion body.
[0038] 2 Fermentation of engineered bacteria
[0039] Draw the line of the preserved engineered bacteria on the kana-resistant LB medium plate, activate overnight at 37°C, pick c...
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The invention relates to a method for separating and purifying a recombinant porcine circovirus capsid protein inclusion body. The method for separating and purifying the recombinant porcine circovirus capsid protein inclusion body comprises that after an inclusion body is produced through induction, an optimized splitting decomposition method is adopted for crushing thalli to primarily remove a part of impurities, and then a series treatment is carried out on the inclusion body, so that the inclusion body forms the soluble target protein. By adopting the method, denatured dissolution and renaturation of the used urea, guanidine hydrochloride and the like on the inclusion body are avoided; meanwhile, nickel column affinity chromatography is carried out, so that yield of protein obtained through purification is high, and the advantages of high purity and simple operation are realized. The method for separating and purifying the recombinant porcine circovirus capsid protein inclusion body has the advantages that the soluble target protein is separated from the insoluble inclusion body and is purified, a tedious step of repeatedly carrying out denaturation and renaturation and danger of protein precipitation in a renaturation process are avoided, and the obtained protein can be electrophoretically pure.
Description
technical field [0001] The invention relates to a method for separating and purifying recombinant porcine circovirus capsid protein inclusion body. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes multisystemic wasting syndrome (PMWS) in weaned piglets. Since the first occurrence of PMWS in Canada in 1991, the disease has been reported in most pig-raising countries in the world, causing significant economic losses to the pig industry worldwide. Among the 11 open reading frames (ORFs) in PCV2 genome, ORF2 is the main open reading frame. In PCV2, the ORF2 start codon starts at the 1033rd or 1034th nucleotide, with a total of 702 bases, encoding 234 amino acids (the ORF2 terminator on the antisense strand of the gene sequence changes from TAA to AAG, resulting in ORF2 The reading frame is extended, the full length is 705bp, and its terminator is TGA), encoding the main functional protein with a molecular weight of about 30kDa—Cap...
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IPC IPC(8): C12P21/02C07K14/01C07K1/22C12R1/19
Inventor 吕暾王立波乔春晓王伟东庄星来洪晶林拱阳陈晟生
Owner FUZHOU UNIV