Study On The Construction And Immunity Of PRRS DNA Vaccine Based On The Alphavirus Replicon

Porcine reproductive and respiratory syndrome characterized by reproductive failures in late-termpregnant sows and respiratory problems in piglets and growing pigs and it has devastated the swineindustry and costed.Currently,both modified-live or inactivated PRRSV vaccine have inherentdrawbacks. Thus, how to develop safer and more effective vaccines remains a challenge.More recently, RNA replicon vaccines have emerged as an important strategy to alleviate theconcerns for potential chromosomal integration and cell transformation generated by the use ofconventional DNA vaccines. We developed our vaccine using the Semliki Forest virus suicidal DNAvector, pSCA1. These vectors do not raise the concern associated with naked DNA vaccines ofintegration into the host genome. This feature is particularly important for vaccine developmenttargeting proteins.In the study, ORF3,ORF5and ORF6gene were amplified by RT-PCR from the cDNA of PRRSVFZ06A, sequenced and modified. then were cloned into pSCA1. The recombinant plasmids werechecked by restriction enzyme analysis and nucleic acid sequencing, and right constructed plasmid werenamed pSCA-Km5, pSCA-VPm3, pSCA-VPm5, pSCA-VP6and pSCA-V56.Then the recombinantplasmids were transfected into BHK-21cells by LipofectamineTM2000reagent. The genes and proteinsof PRRSV expressed were confirmed by RT-PCR, IFA and western blot. After transfention we detectedthe apoptosis cells by fluorescence microscopy. The results of RT-PCR, IFA and western blot suggestedthat the target proteins were expressed well by pSCA1. The recombined plasmid could induce BHK-21cells to apoptosis, which means the recombined plasmids based on pSCA1, are safe.For the detecting later,we cloned the PRRSV GP3,GP5and M protein in the prokaryotic expressionsystem and preparated the polyclonal antibody against M and monoclonal antibodies against GP5.The immunogenicity of five plasmids was evaluated in BALB/c mouse model. For the group ofpSCA-VPm3、pSCA-VP6, specific lymphoproliferative responses to the PRRSV stimulation wereinduced in the splenocytes of the immunized mice as demonstrated by MTS staining assay, and antigenspecific IFN-γ was detected in the splenocytes by cytokine ELSIA. For the pSCA-Km5, pSCA-VPm5and pSCA-VPm5-PEI, low-level of specific antibody was detected by ELSIA, and antigen specific IL-4was detected in the splenocytes by cytokine ELSIA. While, the antibody and IL-4level of thepSCA-Km5is lower than the pSCA-VPm5, which indicated that VP22gene have transductioncapability. The antibody titer of pSCA-VPm5-PEI decreased after7W, which may because of its toxityto cells. No neutralizing antibody was detected in the mice.To sum up, we expressed and purified recombinant m3JD, m5and MJD proteins successfully. Wesuccessfully constructed alphavirus replicon vaccine pSCA-Km5, pSCA-VPm3, pSCA-VPm5,pSCA-VP6and pSCA-V56, based on the vector, pSCA1. And we also did the research about expressioncharacteristics and the immune responses against PRRS in mice of the vaccines.