Porcine Circovirus Disease Genetic Engineering Subunit Vaccine Based On Bacterial Ghosts

Porcine circovirus 2(PCV2)is the causative agent of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD).PCV2 has two major open reading frames(ORF)and the ORF2 encodes capsid protein,which is the structural protein and has good immunogenicity.So Cap protein can serve as target protein to develop newly subunit vaccine.Bacterial ghost is empty bacteria devoid of cytoplasmic contents.On one hand,BG can serve as adjuvant to enhance immune response,on the other hand,BG can be used as delivery system to present foreign protein to antigen presenting cells.In this study,we anchor Cap protein on the outer membrane and delivered Cap protein into the periplasmic space of BG.In addition,we also evaluate immune effect.To evaluate the immune effect of Cap delivered on the outer membrane and periplasmic space,we clone Cap gene downstream GeneIII and INP signal sequence.Then we clone the lytic gene E BOX into the same vector,named as pMD-GKB-cap-E and pMD-28a-cap-E,and transform them into E.coli BL21(DE3)competent cell.The results showed that Cap protein delivered on the outer membrane and periplasmic space can be efficiently expressed.Then we vaccine 6-week old pigs with BL21(pMD-GKBCap-E)and BL21(pMD-28a-Cap-E).Antibody tilers in all the vaccinated group are much higher than control group by ELISA(>1:320).Results show that Cap protein can be well presented by BG and it has good immunogenicity.BL21(pMD-28a-Cap-E)ghost vaccinated group didn’t show viremia by PCR.However,viremia were detected in pigs of control group from second week to forth week post challenge.The virus loads in the inguinal lymph nodes of vaccinated group are much lower(p<0.05)than control group 28 days after challenge;meanwhile,weight growth rates of pigs in vaccinated group are higher than control group(p<0.05).In conclusion,BG can present Cap well and can protect challenged pigs.To evaluate the efficacy of APP ghost as adjuvant of Cap subunit vaccine,this study expressed and purified the soluble recombinant rCap in BL21(DE3).Experimental animals were divided into 4 groups,including APP ghost group,ISA61 group,PCV2 killed vaccine group and control group.The results showed that the APP ghost adjuvant can induce higher antibody tilers than ISA61 group(p<0.05);viremia were not detected in pigs in APP ghost group after challenge,whereas viremia were detected in the pigs of control group from second week to forth week after challenge;the virus loads in the inguinal lymph nodes of APP ghost group are much lower(p<0.05)than control group and ISA 61 group 28 days after challenge;meanwhile,weight growth rates of pigs in APP ghost group are higher than control group(p<0.05).In conclusion,APP ghost can serve as good adjuvant to enhance animal immunity and increase the ability of anti-infection.To produce PCV2–APP bigeminal vaccine,we try to present Cap protein in APP ghost.At first,we clone expression element of delivery plasmid from pMD-19 T to shuttle vector p LS88.Then we transform two recombinant plasmids to S8 strain of APP.However,we didn’t detect the expression of Cap protein by SDS-PAGE and western blot.We speculate the ribosome in the APP can’t recognize the promoter of expression vector.Then we clone olmA,sodc and T4 promoter to the two expression plasimid.We still haven’t detected Cap protein by SDS-PAGE and western blot may resulting from the small quantity of Cap protein.