Immune Protection Of Enhancing DNA Vaccine Of E. Acervulina Hsp90 Gene

Chicken coccidiosis, a kind of cytoplastic entozoic parasite disease, is caused by one or several species of coccidian belonging to genus Eimeria with typical pathological damages in intestine. The development and multiplication of coccidia could lead to diarrhea, bloody stool and decreased weight gain and had serious economic consequences jmpacting the poltry industry. Currently, chemotherapy is used extensively to control the disease, but drug resistance among parasite strains and drug residue has stimulated research interest in the development of alternative control methods. Now, coccidiosis vaccine, especially the genetic engineering vaccine, becomes an alternative approach against the disease. However, Eimeria parasites have complex developmental life cycle, it becomes critical for selecting optimal immune antigen for coccidian. Hsp90 family is an evolutionarily conserved protein that have molecular weight 81~99 Ku, these molecular chaperone proteins play an essential role in stress tolerance, cell cycle regulation, and antigen presentation and exerts important functions in its infection, growth and development.In this study, we cloned E. acervulina Hsp90 gene of 2 433 bp size by RT-PCR from Eimeria acervulina sporozoites. DNAStar analysis showed the open reading frame(ORF) was 2 139 bp, ecoded 713 amino acids and had a molecular mass of 82.5 Ku with isoelectric point of 5.03. We constructed the recombinant plasmid p ET30a- Hsp90 then transformed into E. coli Rosetta cell for induction with IPTG. The recombinant Hsp90 protein was expessed in supernatant and had molecular weight of 89 Ku. The purified recombinant protein as an antigen was used to immunize the rabbit and prepare anti-Hsp90 polyclonal antibody. Indirect ELISA and Western-blot assays showed the antibody has highly reactivity and specialy.A flexible linker(G4S)3 polypeptide was used to link the Hsp90 and Ch IL-18, and consturcted successfully the recombinant eukaryotic expression plasmid of p VAX-Hsp90 and p VAX-IL18-Hsp90, respectively. In vitro transfection experiment with demonstrated that Hsp90 and IL-18 have been expressed. This indicated the fusion gene could be expressed and used as immunoantigen for the following immunization research in vivo. Five groups of chickens were used to evaluate the immune protection efficacy of the recombinet eukaryotic expression plasmid. They are nonimmune and nonifected group(N group), PBS group(P group), p VAX1 group(E group), p VAX-Hsp90 group(H group), and p VAX-IL18-Hsp90 group(I group). Every chicken in different groups was separantely vaccinated with 100 μg of p VAX, p VAX-Hsp90, p VAX-IL18-Hsp90 or 100 μL PBS at day 14 and day 21 respectively, and then challenged with 1×105 E. acervulina sporolated occysts at day 28. The lymphocyte proliferative function, Ig G antibody, the body weight gain, lesion score of duodenum, oocyst per gram(OPG) and anti-coccidia index(ACI) were evaluated. The results showed that the H group and I group has higher protection level than other groups. The ACI in group H and I was respectively 166.17 and 175.32, all more than 160, which indicated Hsp90 could act as good immunoantigen candidate and Ch IL-18 could exert its function as adjuvant. In general, the DNA vaccine could induce good immune protection, and this would provide theoretical and experimental basis for profound study the biofunction of Hsp90 and the immunoprotection of avian coccidiosis.