Research On DNA Vaccine Of Chicken Eimeria Acervulina

The research to immunizition of the Coccidian was given more and more attention in recent 10 years. Founding the antigens that have better immunity is the precondition to give research on theirs recombinant vaccine and DNA vaccine. Tubulin is one of the important organelles of coccidia, which plays an important role during invading into the cells of host. We focus on the research of a -tubulin and did the tests as follows:1.Clone of the E.acervulina a -tubulin gene The total RNA of the sporulated oocysts of E.acervulina BJ strain was extracted and used as the template for RT-PCR. The primers were designed according to the published a -tubulin sequence on Genebank (Yamage M,1995). The BamHI and EcoR I enzyme cleavage sites were added respectively on the upper and lower primer. The amplified PCR product was linked with the pGEM-T vector, and then transformed into the JM109 competent cell, screened through the blue and white spot, identified by PCR and cleaved with endonucleases, and then the positive clone was sequenced. The results indicated that the amplified gene is 1955bp, the homology is 97.4% between the cloned gene and the published gene on Genebank. Now the gene has been published into Genebank as the alpha-tubulin of E.acervulina BJ strain, the published number is AY488134.2.Expression and identification of the a -tubulin gene The expressed primers were designed anew, The ORF sequence of a -tubulin gene was cloned with the positive plasmid as template. Then the ORF gene was linked with pGEX-6P-l vector and transformed into E.coli BL21(DE3), 1.0mM IPTG was used to induce the expression. The SDS-PAGE result indicated that the protein with the molecular weight 69kDa could be expressed to a high level after being induced 2h, and the level arrived to peak 5h later. The expressed protein accounted for 33% in the protein of bacteria. The specific band was identified with GST antibody through western-blot analysis.3.Construction of the eukaryotic vector (p- a -tubulin) of a -tubulin The DNA vaccine p-a -tubulin was constructed by inserting ORF sequence of a-tubulin into eukaryotic vector pcDNA3.1. The plasmid was identified to be positive plasmid by Enzyme cleavage and PCR.4.Research on the protective effect of a-tubulin DNA vaccine and genetic engineering subunit vaccine Both the a-tubulin DNA vaccine and genetic engineering subunit vaccine were set up three doses groups which were high dose(100 u g), middle dose(50 ug) and low dose(25 u g). At the same time, the Diclazuril group, ooyst group, challenged group and unchallenged group were set up, too. The chicken were immunized respectively on the 7th and 14th day of age and challenged with 12×104sporulated oocysts of E.acervulina a week later. Results indicated that the DNA vaccine that used in middle dose reduced the oocysts output 34.38% to the chicken challenged with E.acervulina, the lesion scores of intestine decreased 33.03%, at the same time, the weight gains were obvious, relative weight gain ratio was 82.84%. ACI was 164. All of the indexes were better than the high dose and low dose of DNA vaccine gourps. However, the protective effects of all doses of genetic engineering subunit vaccine groups were lower than the protective effects of DNA vaccine groups.