| Coccidiosis is a kind of intestinal parasitic diseases caused by seven Eimeria coccidia and E.acervulina is one of these major pathogens. The invasion of chicken intestinal epithelial cells by coccidian sporozoites, it makes serious damage to chicken digestive system and resulting in decreased feed conversion ratio and reduced weight gain. It brings great burden to the livestock production. In the past, prevention and treatment of coccidiosis depended on medication of anti-coccidial drug, and it caused serious problem such as drug residues in animal product and drug-resistant coccidia strains. Now, vaccination gradually replaces drug utilization. Screening effective immune antigen is the foundation and key to develop new efficient anti-coccidiosis vaccine. Rhomboid belongs to serine protease family, widely distributes in parasite and performs a variety of functions in the development and signal transduction, particularly in the invasion process of coccidia.Specific primers were designed according to Rhomboid gene sequence(DQ323509.1)published in GenBank. Using E. acervulina cDNA as template, interested gene fragment containing the Rhomboid gene ORF was amplified. After antigenic epitope analysis, specific primers for protein expression were used to amplify the truncated Rhomboid gene. Then it was ligated to pGEX-6P-1 vector and constructed prokaryotic expression plasmid of pGEX-Rhomboid successfully. The plasmid was transfected into E. coli Rosetta and inducted with IPTG. SDS-PAGE detected the expression of expected protein with the molecular weight of 36 ku. After purification,it was used to immunize New Zealand white rabbits for preparation polyclonal antibody. The antibody titer was up to 216 detected by ELISA. Western-blot showed that polyclonal antibody had good reactivity and specificity.Recombinant eukaryotic expression plasmids of pc DNA-Rhomboid and pc DNA-IFN-γ-Rhomboid were constructed. In vitro transfection experiment demonstrated that Rhomboid and IFN-γ have been expressed in BHK21 cells respectively, and could be used as DNA vaccines for the following chicken immune protection experiment in vivo. Five groups of chickens were divided to evaluate the immune protection efficacy of the recombinant eukaryotic expression plasmids. There are two eukaryotic plasmids immune groups of pc DNA-Rhomboid(Rho group)and pcDNA-IFN-γ-Rhomboid(IFN-γ-Rho group), and two challenge control groups of PBS group(PBS group) and pc DNA3.1 group(pc DNA group), and no challenge no immune group control group(Control group). Chicken in different groups was separately vaccinated with 100 μg ofpc DNA3.1, pc DNA-Rhomboid, pc DNA-IFN-γ-Rhomboid or 100 μL PBS at day 14 and day21 respectively, and then challenged with 1×105 E. acervulina sporolated occysts at day 28 except the control group. The lymphocyte proliferative function, IgG antibody, the body weight gain were detected at day 14, 21, 28, 35 and 42, and lesion score of duodenum, oocyst per gram(OPG) and anti-coccidia index(ACI) were evaluated at day 35. The results showed that the T and B lymphocyte proliferative function and specific antibody against Rhomboid in chickens of Rho group and IFN-γ-Rho group were significantly or very significantly higher compared with those of pc DNA group and control group. The ACI of Rho group and IFN-γ-Rho group was respectively166.35 and 172.40, and showed very good anti-coccidial effect. All above, Rhomboid could act as good immunoantigen candidate for coccidian and Ch IFN-γ exerted its function as adjuvant. This study provides theoretical and material basis for research and development of anti-coccidiosis vaccine. |
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