Construction And Immunogenicity Study Of A Bivalent DNA Vaccine Based On PoRV VP7 And PEDV S Gene

Porcine Rotavirus disease is caused by porcine rotavirus (PoRV), which mainly causes piglet diarrhea, vomiting and dehydration. Porcine epidemic diarrhea is a highly contacting intestinal infectious caused by porcine epidemic diarrhea virus (PEDV), which leads to a high mortality rate of piglets. Two viruses often cause mixed infection on piglets, resulting in greatly increasing the fatality rates, which brings huge economic losses to the pig industry. Currently, there is no specific cure for the treatments of viral infections described above. In order to prevent and control the diseases, safely and efficient vaccine researches are the primary solution.1 Cloning and bioinformatics analysis of PoRV VP7 and PEDV S geneThe primers VP7-a/VP7-b and S-a/S-b were separately designed based on the complete genes of PoRV OSU strain (Accession No. KJ450849) and PEDV CV777 strain (AF353511) from Genbank. The fragments of 986 bp and 989 bp were separately amplified from PoRV SC-R strain and PEDV SC-P strain. The target genes were connected to pMD19-T (Simple) and the recombinant plasmids were transformed to DH5a, respectively. With PCR and enzyme digestion identification, PMD-VP7 and PMD-S recombinant plasmids were obtained.The nucleotide identities and phylogenetic analysis were executed with MEGA 5.0, MegaAlign and DNAStar, which showed that the homologies of PoRV VP7 gene were 94.9%-98.7% among SC-R strain and 32 worldwide strains, and 94.7%-98.8% of PEDV S gene among SC-P strain and 38 worldwide strains. The Physico-chemical Properties, Hydrophobicity, Transmembrane domain, Signal peptide, Phosphorylation site, Glycosylation site. Antigenic determinant site, Secondary structure and Tertiary Structure of target proteins were analyzed with kinds of biology softwares. The results showed that PoRV and PEDV target genes amplified in this study both had good immunogenicity.2 Construction of eukaryotic expression vector pPI-2.EGFP.VP7.SPMD-VP7 and pPI-2.EGFP plasmids were digested by EcoR Ⅰ and Kpn Ⅰ. The target fragments were purified with gel extraction kit and transformed to DH5a after the connection reactions. With PCR and enzyme digestion identification, pPI-2.EGFP.VP7 recombinant plasmids were obtained. PMD-S and pPI-2.EGFP.VP7 plasmids were digested by Kpn I and BamH I. The target fragments were purified with gel extraction kit and transformed to DH5a after the connection reactions. With PCR and enzyme digestion identification, pPI-2.EGFP.VP7.S recombinant plasmids were obtained.3 Expression assay of pPI-2.EGFP.VP7.S in vitropPI-2.PEGFP.VP7.S recombinant plasmids were transfected into BHK-21 cells, and the expression of EGFP was observed with inverted fluorescence microscope after 24 h. Total RNA was extracted using TRIzol (Invitrogen) from BHK-21 cells transfected with PPI-2.EGFP.VP7.S for 48 h. The fragments of VP7, S and VP7-S were respectively amplified with RT-PCR, which showed that pPI-2.EGFP.VP7.S had successfully transcribed in BHK-21 cells. Cell samples transfected for 60 h were used to carry out for western blotting. The result showed that the fusion protein has successfully expressed in BHK-21 cells and its molecular weight was about 98 KDa, which had good immunogenicity.4 The dynamic distribution and security study of pPI-2.EGFP.VP7.S in micepPI-2.EGFP.VP7.S was immunized with 100μg per mouse by intramuscular injection, and total DNA was extracted from different tissues in different periods and amplified by RT-PCR method. The results showed that pPI-2.EGFP.VP7.S vaccine reached the sites of heart, liver, lung, spleen, kidney, brain and blood in the mice immunized 3 h later, which stayed in the brain for over 42 d, in the heart and blood for over 63 d, and in the muscle parts of injection for over 91 d. In addition, total DNA was extracted and purified from different tissues in different periods. And VP7-S gene were not amplified from the purified samples, indicating that pPI-2.EGFP.VP7.S in animals had better security.5 Immunogenicity study of the bivalent DNA vaccine in miceTo evaluate the immune effect of pPI-2.EGFP.VP7.S, the recombinant plasmids were immunized with different doses, different approaches and different adjuvant in mice, respectively. The serum samples were collected for PoRV and PEDV IgG antibodies detection at 0 d,7 d,14 d,21 d,28 d and 42 d. The serum samples collected at 42 d were detected on IFN-y and IL-4. The spleens were collected for spleen lymphocyte proliferation test at 0 d,14 d,28 d and 42 d.The immune effect of the intramuscular injection group with 200 μg each mouse was better than the intramuscular injection ones of 100 μg and 50 μg. The immune effect of intramuscular injection group with 100 μg was better than the subcutaneous injection group of 100 μg. The immune effect of IFN-a adjuvant group with 100 μg was better than the intramuscular injection of 100 μg. And the immune effects of transfer factor and IL-12 adjuvant group with 100 μg were a little better than the intramuscular injection of 100 μg.