Expression Of ORF2 Gene Of Procine Circovirus Type 2 In Baculovirus Expression System And Its Immune Effect To Mice As Subunit Vaccine

Porcine circovirus (PCV2) was considered being not only the main causative agent of post-weaning multi-systemic wasting syndrome ( PMWS ) , but also close association with some diseases such as porcine dermatitis nephropathy syndrome( PDNS), porcine respiratory disease complex ( PRDC) , proliferative and necrotizing pneumonia( PNP), congenital tremor (CT) and reproductive disturbance . At present PCV2 has became one of the most important cause in herd, caused huge economic loss in the pig industry, it had widely spread in the world and maintain high infection rate, cause disorder of immune function and lead to the infection of the other disease. In 2000 PCV2 was first found in our country, now the infection rate in our country keep increasing, it has seriously affected to the development of pig industry.Since the virus titer in cell culture is very low, which make it difficult to develop the traditional vaccine such as inactivated and attenuated vaccine,It has become a focus to develop safe effective and cheap vaccines by genetic engineering methods. In the Genome of PCV2, the Cap protein coded by ORF2 gene is the major structural protein, the cap protein has good immunogenicity, so ORF2 gene of PCV2 is the focus on vaccine of genetic engineering. In this study, using insect baculovirus expression system to express the Cap protein, and introducing Kozak sequence to exogenous gene, in order to improve expression of the Cap protein, this study made a basis to the Cap protein as subunit vaccine.According to published PCV2 sequence (AY686764) in Genbank, a pair of primers was designed and synthesized, amplify ORF2 gene and its outer region by PCR, sequence results is Consistent with AY686764. According to sequence results, there pair of primers was designed and synthesized to ORF2, ORF2 gene was amplified respectively by PCR, and clone into the corresponding sites of baeulovirus transfer vector pFastBacΙ, pFastBac ORF2 was transformed into E.coli DH10Bac, then get the recombinant bacmid. The recombinant bacmid was transfect into insect cells Sf9, get the recombinant baculovirus, named VBac-P22, VBac-KP22, and VBac-KGP22.By the identification of IFA, SDS-PAGE and western-blotting, demonstrated that VBac-P22, VBac-KP22 and VBac-KGP22 can express correctly the target protein into insect cells sf9; The result of ELISA antibody test show that: all kinds of proteins expressed as subunit vaccine can stimulate the production of specific antibodies in mice, demonstrated that the target protein has good immunogenicity; The result of detection of protein expression show that: three kinds of recombinant virus infected insect cells sf9 of the same culture conditions, expression level of VBac-KP22 is 0.91mg/108cells , expression level of VBac-KGP22 is 0.56mg/108cells , expression levels of VBac-P22 is 0.24mg/108cells , demonstrated that the introduction of kozak sequence can improve expression level of the recombinant baculovirus to the Cap protein.