| According to the gene sequences of E. acervulina lactate dehydrogenase (LDH) published in the GenBank, we designed a pair of primers and cloned part LDH gene of Shanghai strain of E. acervulina successfully, its size is993bp. Prokaryotic expression plasmid of LDH was constructed, and the expressed recombinant protein is42kDa of molecular weight induced with IPTG. Polyclonal antibody was prepared by immunize New Zealand white rabbit with purified LDH protein. Western-blot and indirect immunofluorescence test indicated that the polyclonal antibody has good reactivity. Using flexible amino acid linker of (G4S)3, we successfully constructed a fusion gene eukaryotic expression plasmid of PVAX-IL-18-linker-LDH. The transiently transfection test showed the fusion expression protein can be detected by LDH and ChIL-18polyclonal antibody, which indicated that the recombinant fusion protein in transfected cells has the biological activity of LDH and ChIL-18, respectively.In animal immunization experiments, we designed five treatment groups, ie. group B (control group), group H (only inoculated with coccidia), group P (inoculated with coccidia and PVAX vector), L group (inoculated with coccidia and PVAX-LDH vaccine), group LK (inoculated with coccidia and PVAX-IL-18-linker-LDH vaccine). During the experiment, chickens were treated correspondingly2times at21and28day-old, and challenge with5×104sporulated oocysts at35day-old. Samples were prepared and assayed at21,28,35,42,49day-old. The results showed that T lymphocyte proliferation function of two immune group of chicken had significantly increase on day28. Whereas on day42, it had very significantly increase in immune group compared with those in control groups. It was found that LK group chicken had better performance than that of L group, but it had no obvious difference in statistics. B lymphocyte and LDH protein stimulated lymphocyte proliferation function test showed similar trend to the results of T lymphocytes assay.The relative growth rate, fecal oocyst number, intestinal lesion score and immune protection efficiency of different immunization groups were compared after inoculated with coccidia. The results showed that there was no significant difference of the average weight gain post immunization between immunized and control groups, which indicated that DNA vaccine had no adverse effects on weight gain. Obvious difference could be found in the average weight gain post challenge, it was significant higher of the2immunized groups than those of group H and P. The oocyst count results demonstrated that there were54.11%and60.44%reduction in group L and LK chickens compared with that of group H. The anticoccidial index was152of group P,176of group L,179of group LK, respectively. It indicated a synergistic effect of cytokine-antigen immune protection.In summary, ChIL-18gene could increase the immune resistance against Eimeria acervulina challenge stimulated by LDH gene DNA vaccine. This study could provide a foundation for further study of the biological activity of LDH, as well as explore the development of novel immune-enhancing anticoccidial DNA vaccine. |
PDF Full Text Request