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

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...

AI Technical Summary

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 in the Bac-to-Bac baculovirus high-efficiency expression system is used to construct the recombinant baculovirus expressing the Cap protein, add the His tag, and use the NI-NTA purification resin to purify the Cap protein, and the obtained pig Circovirus type 2 Cap protein maintains its natural activity and is highly expressed

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