The Study On Nucleocapsid Protein Carrying Label Of Porcine Circovirus Type 2 Expressed By Recombinant Baculovirus

Porcine circovirus(PCV)is a small,non-enveloped,spherical single-stranded DNA virus,and its particle is about 17 nm.PCV is classified into Porcine Circovirus Type 1(PCV1),Porcine Circovirus Type 2(PCV2),and Porcine Circovirus Type 3(PCV3).Among them,a series of diseases related to PCV2,such as Postweaning Multisystemic Wasting Syndrome(PMWS),are called PCVD.PCVD has caused great harm to the pig breeding,and vaccination is still primary means to prevent and control PCV2.The PCV2 vaccines in domestic are mainly inactivated vaccines,the price is on high side,and it is difficult to completely remove infected PCV2 in pigs.The induced antibodies can't be distinguished from wild-type antibodies.Therefore,developing a highly effective and cheap subunit vaccine which the induced antibodies can be distinguished from wild-type antibodies is very important.The PCV2 nucleocapsid protein which is constituted by ORF2 code 233 or 234 amino acid residues of PCV2 is the only structural protein of PCV2,and it's also a major immunogenic protein(Mankertz A et al.,1998).Now,the nucleocapsid protein has been developed into a PCV2 subunit vaccine.This study is devoted to constructing a recombinant baculovirus which can express PCV2 nucleocapsid protein carrying label.And the obtained marker protein can provide material for the study of the PCV2 subunit vaccine:1.The bioinformatics analysis of PCV2 GZ-RH1 strain ORF2 and introduction of Flag epitope tag.To understand the molecular characteristics of PCV2 GZ-RH1 strain ORF2 and to construct PCV2 whole-genome cloning plasmid carrying the Flag epitope tag,after PCV2ORF2 is analysed by bioinformatics software in this study,we have designed special primer,and introduce Flag epitope tag to the terminal of PCV2 ORF2 through techniques such as PCR amplification,TA cloning and so on.PCV2 ORF2 genetic evolution tree shows that the distribution of time and regional of PCV2 is not obvious.And the genotype of PCV2 GZ-RH1 strain belongs to the PCV2 b subtype,the ORF2 is more conservative.The results of the proteinderived from The PCV2 GZ-RH1 strain ORF2 show that ORF2 encoded protein is hydrophilic and mixed protein,its potential phosphorylation sites are significantly different from the existing vaccine strains,while the protein structure is similar to vaccine strain.The ORF2 coding amino acid has an arginine-rich region.The codon bias of the PCV2 ORF2 gene is weak,and the codon bias of this gene is mainly affected by the natural selection pressure.Through Codon usage frequency,it is analysed that when the ORF2 gene is expresses by selective expression system,the insect cell-baculovirus expression system(IC-BEVS)is ideal.The results of plasmid PCR,double enzyme digestion,and sequencing analysis show that the Flag tag is successfully introduced into the C-terminus of ORF2 of PCV2 GZ-RH1 strain,thereby constructing pMD19-T-PCV2-Flag recombinant plasmid.In this study,the ORF2 gene of PCV2 GZ-RH1 strain and its encoded protein are further analyzed and analyzed in depth,which provide a theoretical basis for subsequent studies.The constructed pMD19-T-PCV2-Flag plasmid provide material for recombinant insect baculovirus vector capable of expressing PCV2 Cap marker protein.2.Construction of recombinant baculovirus expression vector of porcine circovirus type 2capsid protein.To construct a recombinant insect baculovirus vector capable of expressing the labeled PCV2 Cap protein,ORF2 subclones carrying labels on pMD19-T-PCV2-flag and pMD19-T-PCV2-V5 plasmids are transferred to the vector pFastBacHTA respectively.The recombinant transfer vector is transformed into DH10 Bac competent cells,and the recombinant bacmid is transfected into Sf-9 insect cells.Rescue of recombinant baculovirus in Sf-9 insect cells is observed by transmission electron microscope,and IC-BEVS expressed marker protein is detected through indirect immunofluorescence antibody(IFA)assay,SDS-PAGE,Western-blotting,etc.The results show that the pFastBacHTA-ORF2-V5 and pFastBacHTA-ORF2-Flag recombinant transfer vectors are successfully constructed and the recombinant bacmid rBacmid-Cap-V5 and rBacmid-Cap-Flag are generated in DH10 Bac competent cells.The recombinant baculovirus rAcMNPV-Cap-V5 and rAcMNPV-Cap-Flag are rescued in Sf-9 cells.The specific fluorescence in the cells is observed by the IFA assay.The target band around 30 kDa is visualized by SDS-PAGE and Western-blotting,which indicatingthat the Cap marker protein can be expressed in the insect cell Sf-9.It also shows that the labeled recombinant protein expressed by IC-BEVS has good reactogenicity to V5 antibody and Flag antibody.This study can lay a solid foundation for the development of the PCV2 genetic engineering subunit tag vaccine and the establishment of supporting differential diagnostic methods.