Porcine Parvovirus: Genome Sequence And Cloning, Expression And Gene Immunity Of Structural Protein Gene

Porcine parvovirus (PPV) causes reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. In this research, the PCR method for the detection of PPV was developed. The genome DNA was sequenced and comparison with those of NADL-2 and Kresse strains were carried out. Then the structural protein gene VP1 and VP2 were cloned, sequenced, expressed and the possibility of DNA vaccine were studied too.A pair of primers was derived from the DNA sequences that have been published. The PCR method was developed for the detection of PPV DNA. The results revealed that the expected 445 bp fragment could be amplified from many kinds of tissues of infected foetus and passaged cells. The amplified fragment was shown to be specific for PPV DNA after digested by EcoRI. This method could detect at least the virus of 10-4 TCID50. 16 positive of 24 samples from clinical sick swine were detected by PCR and only 10 positive were detected by HA test. Theses results showed that the PCR method had the specificity and high sensitivity.On the basis of single PCR of PPV and PRV, multiple PCR was established. The results showed that two specific fragments of 445bp for PPV and 217bp for PRV could be amplified in one PCR. This method could be differentiate PRV infection, PPV infection and mixed infection from reproductive failure.The geneome DNA of PPV has been amplified by two PCR amplifications and the sequence of amplified fragment has been determined. The nucleotide sequence organization of the viral genome was found to be similar to that of the other PPV strains and the homology was high over 98%. The sequence analysis showed that there were two open reading frames (ORFs) in PPV genome. The left ORF had the coding domains for all of NS1 NS2 and NS3. The right ORF had the coding domains for both VP1 and VP2.NS3, NS2 gene were overlapped with NSt gene, VP2 gene were overlapped with VP1 gene.The VP1 and VP2 fragments were amplified from PPV RFDNA by PCR method. Then the products were cloned into pMD18-T vector and the sequence were determined. The homology analysis of these genes with others reported in GenBank showed that these gene were high conserved.The completed VP2 gene were cloned into pET15(b), pET17(b) and pET28(b) vectors respectively and 0.8kb fragment of VP2 gene were cloned into pET17(b) vector, the recombinant plasmid pET15bVP2, pET17bVP2, pET28bVP2 and pET17bVP2f were constructed. Then these plasmids were introduced into E. coli BL21. After induction by IPTG, a high expression was found in products of pET17bVP2f, while no expression protein was detected for other plasmids. These results will be useful for further research of VP2 gene.The completed VP1 and VP2 gene were cloned into the the eukaryotic expression vector of pcDNA 3.1(+) and pCIneo, resulting recombinant expression plasmid pcDNA VP2, pCIneo VP2 and pCIneo VPi. Then these plasmids was transfected into IBRS-2 cells and several clone cells were obtained under the selection of G418. Expressed protein was detected by ELISA. On the basis of above work, these plasmids were used as DNA vaccine to immune mouse. The results showed that all these plasmids could induce specific cell-mediated and humoral immunity. The specific cell-mediated immunity induced by pCIneoVP2 was stronger than that induced by inactived PPV vaccine, the humoral immunity induced by plasmid pCIneoVP1 was stronger than that induced by inactived PPV vaccine. The immune responses induced by co-immunization with pCIneo VP1 and pCIneo VP2 were not stronger than that induced by pCIneo VP2 or pCIneo VP1 alone. This preliminary results showed hope of developing PPV DNA vaccine and worthy to continue further studies.