PCV2ORF4Gene Expression, Antibody Production And Detection Of Its Expression In PCV2-infected PK-15Cell

Objectives Porcine circovirus type2is a primary causative factor of an emerging swinedisease postweaning multisystemic wasting syndrome (PMWS), and clinically characterized bycachexia, dyspnea, and occasional jaundice or pallor in piglets of6-15weeks. PCV2can causeimmune suppression, mixed infection and secondary infection consequently seriously harm tothe swine industry worldwide. It is particularly urgent to reveal the pathogenesis of PCV2infection. In our study, both prokaryotic and eukaryotic expression system were used to expressthe ORF4protein and the purified protein was subcutaneously injected into the rabbits topreparate for the anti ORF4antibody. The titer of polyclonal antibody was then assessed byindirect enzyme-linked immune sorbent assay (ELISA). The prepared antibodies were used todetect the expression of ORF4in wPCV2-infected PK-15cells by immunofluorescence assay.Methods Firstly, a pair of primers (ORF4F and ORF4R) was designed according to theORF4gene sequence and BamHⅠ a ndXhoⅠ r estriction enzyme sitewas added at the end of theprimers. Cell culture-adapted PCV2strain (PCV2-SY) genomes were used as templates forORF4gene amplification. Secondly, the ORF4gene was cloned into the plasmid pGEX-4T-1named pGEX-4T-ORF4. Thirdly, a pair of primers (GST-ORF4F and GST-ORF4R) wasdesigned according to the recombinant vector pGEX-4T-ORF4which contains EcoRⅠ andNotⅠrestriction enzyme site. Then GST-ORF4fragment was amplified and integrated into thevector pPIC9K named pPIC9K-ORF4. To express GST-ORF4proteins, the recombinantplasmids pGEX-4T-ORF4and pPIC9K-GST-ORF4were separately transformed into the BL21and GS115strains. The purified proteins were then injected into the rabbit to raiseanti-GST-ORF4PcAb. Lastly, the confirmed antibodies were used to detect the expression ofORF4in PCV2-infected PK-15cells.Results (1) The recombinant plasmids pGEX-4T-ORF4and pPIC9K-GST-ORF4werecontructed and confirmed by PCR and sequencing.(2) The recombinant plasmids weretransformed into the BL21cells and induced by IPTG. The results of the SDS-PAGE andWestern blot showed that the GST-ORF4protein was successfully expressed.(3) The linearizedpPIC9K-GST-ORF4plasmids were transformed into the GS115cells by electrotransformation.The target protein was also successfully expressed in the BMMY media by methanol inductionand confirmed by SDS-PAGE and Western blot.(4) A titre of the antiserum over1:3000weremeasured by indirect ELISA and the results demonstrated that the antibody was effective.(5)The result of the Western blot was negative in detecting the wPCV2-infected PK-15cells;however the result of the immunofluorescence demonstrated that ORF4protein was discoveredin PK-15cells infected by wPCV2.