Expression And Immune Protective Effecacy Of Rabbits Il-2 Gene And Eimeria Stiedai Rhomboid Gene

Rabbit coccidiosis is a kind of parasitic protozoosis caused by Eimeriidae coccidian(Es), which jeopardizes rabbit breeding industry in our country. Among all rabbit coccidiosis, Eimeria stiedai is the most virulent coccidian. At present, chemical drug is the common method for controlling rabbit coccidiosis, which has created drug-resistant strains and drug residues, so immunological means have become the important research direction for controlling this disease. In this study, the recombinant plasmids were successfully expressed and showed immunogenicity by constructing the recombinant expression vector of IL-2 and Eimeria stiedai Rhomboid gene. Immunized rabbits via multi-point subcutaneous injection were used to analyze the antibody titers, expression levels of IFN-γ and IL-10 in serum. The protective effect of recombinant protein to Eimeria stiedai was evaluated by morbidity and mortality, an average amount of oocysts, liver index, hepatic lesions scoring, and pathological changes in liver. The results are as followed:1. Screening and identification of antigenic domains of Rhomboid gene in Eimeria Stiedai The segmented prokaryotic expression recombinant plasmids of Rhomboid gene of Eimeria Stiedai were constructed, and the target protein was expressed in Escherichia coli. Antigenic domains were screened and identified, western blotting showed that truncated fragment p ET-32a-Rho4 had strong antigenicity.2. Cloning and prokaryotic expression of rabbit IL-2 gene Total RNA was extracted from rabbit lymphocyte, the primers were designed according to the rabbit IL-2 gene in Gen Bank(accession number: AF068057) and the enzyme cutting sites of vector. The open reading frame of rabbit IL-2 gene was amplified by reverse transcription PCR. Recombinant plasmid p ET-32a-IL-2 was constructed and conducted prokaryotic expression, indicating that this plasmid had expected antigenicity.3. Construction and prokaryotic expression of the recombinant plasmid p ET-32a-Rho-IL-2 IL-2 gene and the antigenic domains of Rhomboid gene were linked for co-expression, showing that the p ET-32a-Rho-IL-2 had expected antigenicity.4. Protective test of recombinant protein p ET-32a-Rho-IL-2 to immunize rabbit Healthy rabbits were immunized with recombinant proteins p ET-32a-Rho, p ET-32a-IL-2 and p ET-32a-Rho-IL-2 by multi-point subcutaneous injection. Antibody titers and expression levels of IFN-γ and IL-10 were detected by ELISA during two weeks at after three immunization. The results showed that: antibody titers and the expression levels of IFN-γ, IL-10 of p ET-32a-Rho and p ET-32a-Rho-IL-2 were rasied. Although all immunized and normal rabbits were infected by Eimeria stiedai, the hepatic lesions of the p ET-32a-Rho-IL-2 group were less than that of other groups. Indicaty that the recombinant protein could stimulate the body to produce immune response of Th1 and Th2 at different levels with certain immune protection.