Baculovirus Surface Display Of Orf2 Envelope Glycoprotein Of Porcine Circovirus Type 2 And Immunogenicity In Mouse

Porcine circovirus type 2(PCV-2)is the major pathogen for the post-weaning multisystemic wasting syndrome ( PMWS ) and cause exhaustion and death in post-weaning piglets. PCV-2 also is relevant to congenital tremor (CT),porcine dermatitis and nephropathy syndrome(PDNS) and porcine respiratory disease complex(PRDC). PCV-2 can casue immnosuppression in infected piglets. PCV-2 also co-infected with porcine reproductive and respiratory syndrome virus(PRRSV), Porcine parvovirus(PPV) and Pseudorabies virus(PRV) in pig population and cause immune failure of infected pigs which will be followed by the secondary infetions of other pathogens.Hence we can see that PCV-2 infection has already become one of the major pathogens for the pig industry and can cause immeasurable indirect losses.The genome of PCV-2 is a closed circular single-stranded DNA of about 1 767 bp or 1 768 bp,which consists of two open reading frames(ORF),ORF1 and ORF2.ORF1 encodes virus replication associated protein. ORF2 encodes virus capsid protein(virus structural protein).This structural protein has fairly good immnogenicity,but it has no cross protection between PCV-1 and PCV-2. So a better understanding of the ORF2 proteinof PCV-2 will be very important for the PCV detection and vaccine development.The baculoviruses are a family of large rod-shaped viruses that naturally only infect the insect cells.The attachment and invasion process of the virus are associated with the envelope glycoproteingp64 which consists of N-terminus signal peptide and maturation protein(including transmembrane domain and cytoplasmic domain).gp64 has already been developed as the viral surface display system to display various objective proteins. The exogenous gene segment inserted between the signal peptide and the maturation protein.After the post-transcriptional processing,the signal peptide will be cutted off.The new fusion protein will be express steadily on the surface of the virion. After several times of selection,the recombinant baculoviruses with specific protein being expressed can be obtained.This expression pathway is stable and reliable.It is brief and fast when applied to develop genetically engineering vaccines.This technology has been widely applied to design and develop new genetically engineering vaccines.This sdudy display capsid protein of PCV-2 using baculovirus display system and animal immune experiment about the displayed protein. A conclusions of this study are following:(1)A pair of specific primers was designed( P1/P2). ORF2 gene of PCV-2 was amplified by PCR from PCV-2 YL strains and ligated with pGEM-T vector. The recombinant plasmids were transformed into Escherichia coli (E.coli)DH5α. The positive colonies were screened through the white-blue plaque selection. Then the bacteria were cultured in LB medium.The recombiant plasmid was extracted and identified by PCR and restriction enzyme digestion . The ORF2 gene of PCV-2 YL strains was sequenced and then compared with other PCV-2 strains available on GenBank. The correct plasmid was named as pGEM-T-ORF2. The results showed that amplified fragment was 702 bp,encode 234 amino acids and expressed a 28ku protein. The nucleotide identity and amino acids homology of PCV-2 ORF2 were 89.9%-98% and 88.5%-98.3% with other PCV-2 strains available on GenBank.(2)The ORF2 fragment was obtained from pGEM-T-ORF2 digested with BamHI / XhoI and inserted into plasmid pET-32a digested with tha same enzyme to construct the recombinant expression plasmid pET-32a-ORF2. E.coli competent cell was transformed with pET-32a-ORF2 and induced with IPTG.. The fusion protein was expressed in E.coli and reacted with the positive serum of PCV-2 by Western blot, it showed the expressed protein had fairly good antigenicity.(3)A pair of primer was designed according to the sequences of the eukaryotic expression vector Bac-to-Bac and the ORF2 of Porcine circovirus type 2. The complete ORF2 fragment was amplified from recombinant plasmid pGEM-T-ORF2 by PCR. The purified ORF2 fragment was digested with XhoI and PstI and inserted into the eukaryotic expression vector Bac-to-Bac digested with the same enzymes to construct the recombinant baculovirus BscSC-ORF2. The recombiant capsid protein was effectively expressed on the transfectd insect cell membrane observed with immunofluorescence-confocal microscope. The recombiant virus was obtained by high-speed centrifuge and the recombinant protein was observed on the baculovirus sac membrane by immuno-gold electron microscopy.(4)Animal immune experiments indicated that the recombinant protein could trigger fairly strong immune response.Neutralization test indicated that sera from mice immuned with the recombinant baculoviruses could effectively neutralize PCV-2 protein.Lymphocyte transformation prove experiment indicated that the recombinant baculoriruses could stimulate stronger cell mediated immune respone.PCV-2 capsid protein was expressed using baculorirus display system in this study.The reconbinant baculoriruse displaying PCV-2 capsid protein was successfully constructed and established a fundament for preventing PCV-2 infection with the recombinant baculoriruses. The recombinant baculoriruse can be developed a candidate of subunit vaccine for preventing PCV-2 infection.