Construction And Experimental Immunity Of Recombinant DNA Vaccines Pvax1-orf2Against Mycoplasma Suis

ObjectiveTo construct recombinat DNA vaccines pVAX1-ORF2against Mycoplasmasuis,and test specific antibody IgG in serum, Spleen T lymphocytes and kind ofquantity Th1/Th2cytokines after inoculating C57BL/6mice against therecombinat DNA vaccines pVAX1-ORF2plasmid, which to investigate theexperimental immunity of recombinat DNA vaccines pVAX1-ORF2againstMycoplasma suis.And lay the theoretical basis for further clinical research andproduction of DNA vaccines pVAX1-ORF2against the Mycoplasma suis.MethodsThe gene segment ORF2was synthetized through in vitro total genesynthesis. The product was cloned to the vector of pMD18-T,and sequenced toconstructe eukaryotic expression vector pVAX1-ORF2.C57BL/6mice weredivided randomly into group A (pVAX1-ORF2group),group B (pVAX1group),group C(blank control group). Their tibialis anterior muscles were inoculatedagainst100μg liquid three times,once every1week.The mice were cut tails andthe serum were collected when the0,1,2,3,4weeks after the first inoculation,which to save40C for IgG testing. Getting blood and spleen from decapitation inmice when the4weeks later, which were tested specific antibody IgG in serumand Th1/Th2cytokines by ELISA kits,which were detected Spleen Tlymphocytes and kind of quantity CD4+and CD8+by FCM. Results1.The gene segment ORF2was synthetized through in vitro total genesynthesis.2.The Mycoplasma suis recombinat DNA plamsid pVAX1-ORF2wasconstructed successfully.3.The results of mice serum antibody detection in different time show, thespecific antibody IgG were detected when the recombinat DNA plamsidpVAX1-ORF2were inoculated1week later, the highest point were detectedafter the third inoculation1week later, all points of OD450nmwere hinger than thenegative control group (pVAX1group) and blank control group（P<0.05）. Thesplenic CD4+and CD8+T lymphocytes percentage of recombinat DNA plamsidpVAX1-ORF2group apparently higher than the pVAX1control group and blankcontrol group（P<0.01）, and the ratio of CD4+/CD8+were higher than thecontrol groups(P<0.05). The cytokines IFN-γand IL-4, which were screted byTh1/Th2cells, were higher level expressed（P<0.05）.Conclusions1.The ORF2protein showed regular secondary structure and high antigenicindex.2.The immunogenicity gene segment ORF2was expressed in eukaryoticsystems.3.The results of experimental studies on immunnity of the recombinat DNAvaccines pVAX1-ORF2against Mycoplasma suis show, the recombinat DNAvaccines made mice to product the specific antibody IgG, increased the contentof lymphocyte CD4+and CD8+,induced humoral immunity response and cellularimmune response; The mice inoculated against the recombinat DNA vaccinespVAX1-ORF2secreted high level Th1/Th2cytokines IFN-γand IL-4while werestimulated by specific antigen peptide.