Prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein

Abstract

The invention discloses a prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein. The method comprises the following steps: taking a PCV2 strain genome as a reference sequence, coupling an SUMO solubility-promoting expression tag coding gene at the upstream of a Cap protein coding gene of PCV2b, introducing 6 His tag encoding genes at the upstream of SUMO, performing gene optimization, recombining the gene to a PUC57 plasmid, taking the recombinant plasmid as a template, designing a specific primer, after PCR amplification, cloning the product into a pMAL-C4x prokaryotic expression vector, constructing a recombinant plasmid, transferring the recombinant plasmid into escherichia coli, and performing induction and purification. The method successfully constructs the pMAL-MBP-HisSUMO-Cap recombinant expression vector, MBP-HisSUMO-Cap fusion protein can realize soluble expression in BL21(DE3), after purification, the MBP-HisSUMO-Cap fusion protein can be used asimmunogen and can have a specific reaction with PCV2b positive porcine serum, a rabbit anti-MBP-tag polyclonal antibody and immunized mouse antiserum, and results indicate that the protein has good immunogenicity, and lays a theoretical foundation for development of a PCV2b diagnostic kit.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein. Background technique [0002] Porcine circovirus type 2 (Porcine Circovirus Type 2, PCV2) is a non-enveloped, single-strand, circular, negative-strand DNA virus. ), dermatitis and nephrotic syndrome (PNDS). Clinical features are often necrotizing lymphadenitis, acute pulmonary edema, reproductive impairment, and granulomatous enteritis. In clinical cases, PCV2 is often associated with porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), porcine parvovirus (Porcine parvovirus, PPV), porcine pseudorabies virus (Porcine pseudorabies virus, PRV), classical swine fever (Classical swine fever virus, CSFV), streptococcus (Streptococcus) and Escherichia coli (Escherichia coli) and other pathogens mixed infection, seriou...

AI Technical Summary

Problems solved by technology

Scholars at home and abroad have successively used eukaryotic and baculovirus expression systems to express Cap protein, laying a solid foundation for the research of Cap protein, and Escherichia coli has fast growth speed, fast culture, high expression level, candidate plasmid and host It is a commonly used protein expression system, but foreign proteins are easily degraded by host proteases or form inclusion bodies when they are expressed at a high level in E. coli

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