| Coccidiosis, caused by a variety species of the genus Eimeria, is an important disease which harms modern chicken-keeping and exists widely.E.tenella is an important species among those protozoans. Until now a several problems need to be solved like drug-resistance, residual drug, so immunoprophylaxis has become the study focus of coccidiosis. The genus Eimeria includes nine species and there has antigens variation between species, strains and different developmental stages, making available vaccine not completely protect different strains E.tenella. The molecular biology technique has widespread been applicated in Coccidiosis immunoprophylaxis study, but the most important reason which limits the study of E.tenella is that genome project for E.tenella is not completed. Selection of hybridization parents Single sporocyst from CBG strains and HSC strains, which was separated by micromanipulation technique, were inoculated 1-day-old chicken and five chicken were successfully inoculated. Oocysts were collected respectively and made spores. Their genome DNA was extracted and amplified using random primers. The results demonstrated that CBG7 and CBG11 were two clonal populations which had pathogenicity and immunogenicity. Their protection from themselves rates were 86% and 81% respectively, and their cross protection rates were 65% and 59%. So they were used as hybridization parents in the experiment. Cultivation of hybrid strain F3 Genetic stability of CBG7 and HSC11 strains were analyzed by comparing genomic fingerprints between the passaged and primary strains. Their immunogenicity were analyzed by OPG counting, stool scoring, cecal lesion scoring, BW assay and protection rate calculating when 10-day-old chicken were inoculated. The 1-day-old chicken was inoculated with the mixed oocysts including CBG7 and HSC11 strain to get oocyst and single sporocyst was separated to inoculated chicken, hybrid strain F3 , whose pathogenicity and immunogenicity were analyzed, was established. A special fragment was cloned from CBG strain and it only hybridizated with CBG strain and hybrid strain F3, demonstrating the gene has high specificity. Cloning of gene related to microneme from E.tenella A sequence included thrombospondin and terminator was discovered by sequencing of E.tenella. RAPD sequence and it has structure domain characterized by microneme-protein family which was amplified by 5'-RACE and denominated by EtM32. Analysis of Amino acid sequence and protein function demonstrated that the gene was related to micronemes which are important for parasite motility and host cell invasion. Prokaryotic expression of EtM32 related to microneme The recombinant plasmids pET-EtM32 was constructed by cloning EtM32 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host BL21(DE3).The expression levels were optimized by manipulating host strain,the media,the time of harvest. The recombintant proteins were highly expressed in normal LB media, then induced by the 1mmol/L IPTG for 5h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant EtM32 wre up to 22% of the total bacterial protein in the cell lysate. It was specificity of E.tenella by Western-blotting. Immunoprotection of recombinant antigen against E.tenella challenge The chicken were inoculated with recombinant antigen by different pathways, then were challenged with E.tenella oocysts. The results showed recombinant live E.coli strain could induce immunologic reaction and protect partially against E.tenella challenge. Oocysts number and timing shedding of experiment grops were shorter, the increase of body weight (BW) were swifer, and the cecum Lesion were slighter than those of the control . The studies has established foundations for interpreting immunoprotection... |
PDF Full Text Request