Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus

A pfastbac-p10-orf2-ph-orf2, baculovirus technology, applied in the field of genetic engineering, can solve the problems of low protein expression, insufficient activity and the like

Inactive Publication Date: 2012-12-26
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0010] In order to overcome the defects of low PCV2Cap protein expression and insufficient activity in the existing methods, the purpose of the present invention is to provide a method for expressing porcine circovirus type 2 (PCV2) Cap protein using pFast Bac Dual baculovirus. The pFast Bac Dual baculovirus transfer vector i

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  • Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus
  • Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus
  • Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus

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Embodiment

[0038] A method utilizing pFast Bac Dual baculovirus expression system to express PCV2Cap protein, comprising the following steps:

[0039] 1. Construction of recombinant shuttle vector Bac p10-ORF2-pH-ORF2

[0040] 1. Extraction of PCV2 DNA

[0041] The PCV2 strain (Fan, Ye et al.2012) was inoculated into PCV-free PK15 cells, and the 2 After culturing in an incubator, the cells were collected and processed, and PCV2 DNA was extracted according to the instructions of the OMEGA Blood DNAKit kit as template DNA.

[0042] 2. PCR primer design

[0043] According to the sequence of PCV2ORF2, 2 pairs of specific primers were designed:

[0044] P10F: 5'- CCATGG ATGCACCACCATCATCATCACCACACGTATCCAAGGAGGCGTTT-3';

[0045] P10R: 5'-TGCA GGTACC TTAAGGGTTAAGTGGGGGGTCT-3';

[0046] PHF: 5'- GGATCC ATGCACCACCATCATCATCACCACACGTATCCAAGGAGGCGTTT-3';

[0047] PHR: 5'-CGC AAGCTT TTAAGGGTTAAGTGGGGGGTCTT-3';

[0048] Amplify the ORF2 gene (about 720bp) containing His tag. The four pri...

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Abstract

The invention discloses a method for expression of a PCV 2 (Porcine circovirus type2) Cap protein by a pFast Bac Dual baculovirus. The method comprises the following steps of: amplifying a gene fragment of an encoded PCV 2 Cap protein with a His tag; connecting the gene fragment to a pFast Bac Dual plasmid so as to obtain a recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2; transforming Escherichia coli DH10Bac with the recombinant transfer plasmid, and carrying out blue-white selection to obtain a recombinant shuttle vector Bac-p10-ORF2-pH-ORF2; transfecting an insect cell with the recombinant shuttle vector so as to obtain a recombinant baculovirus Ac.Dual-Cap; and poisoning the insect cell with the recombinant baculovirus, performing cultivation, then collecting the insect cell, and purifying an expression product so as to obtain the recombinant Cap protein. The method disclosed in the invention solves the problem of low expression level of the Cap protein in eukaryotic cells. The recombinant Cap protein in the invention is designed with a His tag, thus being beneficial to the follow-up purification. And the recombinant Cap protein has biological activity superior to that of a Cap protein expressed by a prokaryotic expression system, thus being applicable to establishment of epidemiological diagnosis methods and reseach as well as development of PCV2 subunit vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for expressing porcine circovirus type 2 (PCV2) Cap protein by using pFast Bac Dual baculovirus. Background technique [0002] Porcine circovirus type 2 (PCV2) is an important pathogenic microorganism newly discovered in recent years, which is considered to be the main cause of post-weaning multisystemic wasting syndrome (PMWS). Pathogens (Allan, Meehan et al. 1998). The infection of PCV2 will cause the animal body to produce immunosuppression, reduce the immune resistance of the body, cause secondary infection of other pathogens, and cause more serious clinical symptoms. In addition to causing PMWS, PCV2 can also cause related diseases such as porcine dermatitis and nephropathy sundrome (PDNA) and porcine respiratory disease complex (PRDC) (Allan, McNeilly et al.2000; Kim, Chung et al. 2003; Ramamoorthy and Meng 2009). [0003] PMWS was first discovered...

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Application Information

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IPC IPC(8): C12N15/866C12R1/93
Inventor 樊惠英廖明陈春丽叶昱张杰
Owner SOUTH CHINA AGRI UNIV
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