The Study On Immune-Regulating DNA Vaccine PCDNA-TA4-IL2 Against E Tenella

By technology of DNA recombination,the genes of Chicken IL-2 and, E.tenella antigen TA4 and TA4-IL-2 were cloned into the expression plasmid pET28a and pET28b vector,and expressed in E.coli by IPTG induction. The result of SDS-PAGE shew that the there were proteins with molecular weight about 25kD, 22kD, 40kD respectively which corresponding to pET28b-TA4, pET32a-IL-2 and pET28bTA4-IL-2. After ultrasonicated, these proteins of sentiment were isolated and puried primarily, then renatured. Meanwhile SDS-PAGE also shew that the recombinant protein of ChIL-2 and TA4-IL-2 existed in both sentiment and supernatant But most of the expressed protein of TA4 existed in sentiment, little in supernatant .MTT was applied to detect the ability to stimulate chicken spleen lymphocyte infected with Ktenella of these recombinant proteins of pET28b-TA4 , pET32a-IL-2 and pET28bTA4-BL-2 . The results shew that the recombinant proteins of ChIL-2 , TA4and TA4-EL-2 all could simulate the Chicken spleen lymphocyte profileration after co-culture for 48h and 96h. . The supernatant of pET-TA4-IL-2 was the best one. This was one of important reason for us to select TA4 gene as DNA vaccine candidator and ChIL-2 as immune enhancer.The DNA vaccine of pcDNA 3.1bTA4, pcDNA IL-2, pcDNA-TA4-IL-2 were constructed respectively in the experiment. The three DNA vaccine were injected into chicken chest muscle, then the transcriptions of the genes were detected by reverse transform polymarase chain reaction (RT-PCR) and proteins expression of pcDNA-TA4 and pcDNA-TA4-IL-2 by western blot. The results shew that both mRNA and proteins could be respectively detected in vivo by RT-PCR and Western blot after injecting these DNA vaccines.The recombinant proteui of pET28-TA4, pET28-IL-2, pET28-TA4-IL-2 and the DNA vaccines of pcDNA-TA4, pcDNA-IL-2, pcDNA-TA4-IL-2 were used to immunizeChicken by injecting into chest muscle at age of 17 and 24 day, the chickens werechallenged with Eimeria tenenlla sporalateoocysts at 0.1 million per quota by oral at age of 31 day. The result shew the average body weight gain were significantly different between immunized groups and infected and unimmunized group, but the immunized groups have no difference from the the uninfection and unimmunized group.The relative weight gain ratio of the groups of infected and unimmunized , pET32-IL-2 , pET28-TA4 , pET28-TA4-IL-2, pcDNA-TA4, pcDNA-TA4-IL-2, pcDNA-IL-2 were 68.15%, 96.5%, 87.4%, 81.9%, 90.4%, 97.3%, 93.7% respectively; The total oocysts numerber discharged by infected and unimmunized group was more 2-4 times than the immunized groups,the oocysts decreasing rate of the proteins groups of TA4, IL-2, TA4-IL-2 and DNA vaccines groups of pcDNA-TA4, pcDNA-TA4-IL-2, pcDNA-IL-2 , were 61.1%, 71.6%, 58.1%, 68.7%, 75.1%, 66% respectively; The Lesion score of caeca,of Infected and unimmunized group is markedly different from that of immunized groups and uninfected and unimmunity group, the average score of the groups of infected and unimmunized, uninfected and unimmunized , pET32-IL-2 , pET28-TA4 , pET28-TA4-IL-2,pcDNA-TA4 , pcDNA-TA4-IL-2, pcDNA-IL-2 were 3.5, 0, 1.1, 0.5, 1.3, 0.6, 0.4, 1.6respectively;The overall estimation anticoccidia index of the proteins groups of TA4, IL-2, TA4-IL-2 and DNA vaccines groups of pcDNA-TA4, pcDNA-TA4-IL-2, pcDNA-IL-2 were 185, 182, 177, 183, 192, 176 respectively . We could conclude that the DNA vaccines had the function to protect chicken from coccidian infection. The effectiveness of the pCDANTA4IL-2 is more evident especially.