The Recombinant PPV With Major Antigen Site Of PCV-2Cap Protein And Its Immunogenicity To Piglets

Porcine parvovirus is one of the major pathogens that caused reproductive failure inswine, which is characterized by pregnant gilts and sows occurrence abortion, stillbirths,sterility, embryonic, mummification and weak fetus. Porcine circovirus type2(PCV-2) is theprimary causative agent of postweaning multisystemic wasting syndrome (PMWS). PCV-2has a predilection for lymphoid tissue, therefore replication of the viruses could haveprofound immunomodulating consequences and predispose to debility and secondaryinfections. In the actual production process, PPV and PCV-2often co-infect or secondarilyinfect swine herds, they may interact synergistically to aggravate the development of diseasescaused significant economic loss to swine industry.In this study, the PPV-JT strain was replicated in PK-15cell by synchronous inoculation.This replicative-form (RF-) DNA was obtained and then cloned into pUC18vector to gain theinfectious DNA clone named pPPV-JT, which was sequenced and analyzed. As a result,PPV-JT strain genome containsed4941bp. Nucleotide sequence alignments with relatedreference viruses strains which available from GenBank demonstrated that the NS1gene ofPPV-JT had98.2–99.9%identity among the sequences, the NS2gene97.9-100%, NS3gene98.5-100%, the VP1gene98.6-99.9%, the VP2gene98.3–99.8%,, showed the PPV-JTgenome were comparatively conserved.Phylogenetic analysis showed that the NS1gene of PPV-JT strain had close relationshipwith PPV China strain; the NS2and NS3genes had a close relationship with PPV BQ strain;the VP1gene and VP2gene was closely related to the corresponding genes from NADL-2.The phylogenetic analyses suggested that the PPV-JT strain might be a new recombinant PPV.Compared with NADL-2strain which shown the highest homology with the correspondinggene from PPV-JT strain, there was one non-synonymous substitution in the NS1protein andVP2protein of PPV-JT strain, respectively. It was Trp440Leu in NS1protein. The mutation inVP2protein was Pro304Ser locating in the site4linear epitope, which potentially affected antigenicity and pathogenicity of PPV. The phosphorylation sites of NS1protein of PPV-JTstrain were Thr435and Ser473. The378D,383H,436S of VP2protein which play a role onthe tissue tropism, and two tandem127bp repeats were similar to PPV NADL-2strain, so wededuced that the PPV-JT strain might have the similar virulence with NADL-2strain which isan attenuated strain.As a carrier, PPV had many advantages for the genome of PPV was small, easy tooperate and may effectively expression of foreign genes. The PK-15cells transfected withpPPV-JT mediated by liposome, and then the virus particle was observed, which namedPPV-RF．The physic-chemical identify, specificity experiment and PCR identify showed thatthe rescued virus shared similar properties with the parental virus. This study laid a materialand theoretical foundation for the further research on the recombinant virus with the PPV ascarrier.The276~450gene fragment including the117~131AA linear antigen of ORF2wasamplified from PCV-2strain by PCR, and cloned into the pPPV-JT gene at Nco I position(located at1/3C terminus of VP2) and the recombinant plasmid pPPV-Cap was obtained. Therecombinant plasmids were transfected into PK-15cells using transfection reagent.Common virus identification, molecular biology identification and IFA were carried out.Results indicated that the virus particles named PPV-Cap were obtained and the target geneswere transcripted and expressed correctly.Healthy piglets were immunized to detect the immunogenicity of PPV-Cap.The resultsdemonstrate that the PPV-Cap recombinant virus has good immunogenicity and could inducespecific humoral immunity detected with ELISA. These results provide reference data for thePPV-PCV2recombinant vaccine research.