Construction Of Immune-Enhancing DNA Vaccine Of Chicken Eimeria Acervulina And Its Immune Responses

Coccidiosis is avian parasitic disease, with typical pathological changes in intestine, could cause significant economic losses in the poultry industry. Application of coccidiostats is a main and effective method to prevent coccidiosis, however, drug residue and resistance are the obvious disadvantages of most anticoccidial drugs. Now, coccidiosis vaccine becomes an alternative approach against the disease. The development of immune-enhancing DNA vaccine gradually became the focus of the scientific research.Specific primers for Ea1A cDNA of Eimeria acervulina were designed according to published nucleic acid sequence of Ea1A cDNA of E. acervulina. We amplified a truncated Ea1A by reverse transcription (RT)-PCR, which was 1256 bp in length. The Ea1A and ChIL-18 protein, with MW of 51 KDa and 26 KDa, were induced in E. coli Rosetta system, respectively. After purification by QIAGEN Ni-NTA resin, Ea1A and ChIL-18 protein as immuno-antigens were injected into rabbits to prepare their polyclonal antibody. Cell transfection and immunofluorescence assay and Western blot showed that the polyclonal antibody had good reactivity.In this research, a flexible linker which express the (G4S)3 polypeptide was used to link the 3-1E with Ea1A to construct fusion gene eukaryotic expression plasmid of PVAX-3-1E-linker-Ea1A. The results of transient transfection of the fusion plasmid in vitro and in vivo showed that the fused gene expressed together in BHK cells and could be located by specific anti-3-1E polyclonal antibody and anti-Ea1A polyclonal antibody. Therefore, it proved that the protein expressed by fusion gene could retain the bioactivities of two proteins, it also provided the theoretical foundation for the following immunization research in vivo.The experimental chicken were divided into 7 groups, PBS group inoculated intramuscularly with PBS only, PVAX group with empty PVAX vector, IL-18 group with PVAX-IL-18, 3-1E group with PVAX-3-1E, Ea1A group with PVAX-Ea1A, LK group with PVAX-3-1E-linker-Ea1A and LK+ group with PVAX-3-1E-linker-Ea1A and PVAX-IL-18 combinatively. Thereafter, lymphocyte proliferative assay, enzyme linked immunosorbent assay and index of immune organs were used to detect the immune response of different groups. The results showed that most of immune response of five inoculated groups are higher than the control group, especially, the ChIL-18, LK and LK+ group. The details were as follows: T, B lymphocyte proliferative function, the levels of special antibody against 3-1E and Ea1A in peripheral blood and the index of immune organs were all higher than those of control group. And the LK+ group was the most significant among those groups. Above all, the results of this experiment showed that the host immune responses were improved by inoculated intramuscularly with PVAX-3-1E-linker-Ea1A. ChIL-18 could enhance the immune protection of the host to coccidiosis. Furthermore, 3-1E and Ea1A fusion plasmid was first constructed in this experiment and the results observe here could provide solid scientific ground to the further study of ChIL-18 bioactivities, and the theoretical basis to the discovery of new type immune-enhancing vaccine against coccidiosis.