| Eimeria,an obligate,intracellular protozoan parasite,is the causative agent of coccidiosis.Coccidiosis has become a serious enteroepithelium disease of chicken, it is one of the important diseases which hinder the development of poultry industry. Eimeria has been classified to 9 species according to oocysts characterization, part of parasitism, pathological changes characterization,et al. Eimeria tenella is generally considered to be the parasite species responsible for serious infection in chicken.In our country, Administration of chemical medicines are the main measures for treatment and prevention of coccidiosis.With the occurrence of medicines residual and strains of medicine-resistance coccidia, it is necessary to look for new immunizing measures for prevention and treatment of coccidiosis. Actin depolymerizing factors have been identified from different organisms. These protein appear to regulate actin polymerization in the cell and that its inactivation in yeast is a lethal mutation suggests it plays an essential and fundamental role in regulating actin filament dynamics. Therefore, it is necessary to study E.tenella ADF for regulating motility of coccidia actin, supressing the invasion of coccidia and prevention of coccidiosis. In the present study , ADF recombinant protein was expressed and anti-ADF monoclonal antibody was prepared. the ADF was localizated in E.tenella sporozoite by immunofluorescence and immunohistochemical technique. The recombinant ADF protein could induce protection against E.tenella after chicken were inoculated with it. The results suggested that the ADF gene may be the vaccine candidate gene,This study has established foundations for prevention of coccidiosis.Cloning and expressing of ADF gene Primers were designed according to ADF nucleotide sequence GenBank. The ADF open reading frame(ORF) was amplified by PCR with plasmid contained ADF gene which was preserved in our laboratory. The ADF ORF was cloned into PMD-18T Vector,the ligation product were transformed to competent cells of E.coli host strain DH5α, the positive clones were screened and the positive recombinant plasmids were identified and prepared for automatic sequencing. The results indicated that ORF was 357bp. The nucleotide sequence shared 100% sequence homology with original sequence compared by BLAST. The recombinant expression plamids was constructed by cloning ADF ORF into prokaryotic expression vector pET-28(a) and expressed in E.coli host cells Rosetta (DE3). The molecular weight of the recombinant fusion protein was 17KDa analyzed by PAGE. The expression levers were optimized by adjusting temperature, the concentration of IPTG and the time of harvest. Under the condition of 0.8mM IPTG for 6h , the recombinant proteins were highly expressed in normal LB media. The yield of recombinant ADF (rADF) were up to 40% of the total bacteria protein in the cell lysate. It was analyzed by western blotting.Preparation of monoclonal antibody against ADF Six female BalB/c mice aged 6-8 weeks old were imunized intraperitoneally at day 1,15 and 29 with the emulsive mixture of rADF and CFA/IFA adjuvant. The immunization dose of rADF to one mouse was 120μg.At day 40,serum titers were determined by indirect ELISA. Mice immunized with rADF produced sera exhibiting antibody titers ranged 1:16000 more. Splenocytes from mice immunized with rADF were fused with SP2/0 myeloma cells. Two hybridomas of stable secreting ADF-McAb were selected and were named 2E5 and 2D6。The numbers of chromosome of the two hybridoma cell lines were 88 and 93,respectively. By analyzing the chromosome quantities of the two hybridomas, that is to say the cell fusion were done well. The subclass of 2E5 was IgM, the subclass 2D6 was IgG1.The ELISA titers of 2D6 and 2E5 ascites were 1∶1 .0×10~6 and 1∶1 .0×10~5.Localization of ADF in F2 strains E.tenella sporozoites E.tenella were amplified by oral administration oocysts to 10 days chicken. Sporozoites were Collected by digestion of sporocyst with 0.25% trypsinase and 0.75% sodium taurocholate for 50min. The localization of ADF in E.tenenlla were determined by immunohistochemical and immuno -fluorescence using 2D6 McAb. The results showed that ADF is scattered throughout the sporozoite cytoplasm primarily around the nucleus in the center of sporozoite,but was less at the poles. This result has established foundations for E.tenella ADF further study .The immuoprotection induced by recombinant ADF antigen in chicken The chicken were inoculated with rADF at 7 days,14 days and 28 days old by different ways, then were challenged with E.tenella oocysts. The responses of specific cell immunity were detected by flow cytometry at 7 days after the third immunization. The level of CD4+ lymphocyte and the ratio of CD4+/CD8+ of immunized chicken were increased significantly compared with control groups(P< 0.01).This results showed the rADF can induce the immune response significantly. Other immounoprotective index were detected after chicken were challenged with oocyst. In oral group, oocyst number and cecum lesion were decreased significantly compared with infection control groups(P<0.01),and the increase of body weight (BW) was not significant compared with non infection control group(P>0.05) , but was increased significant when compared with infection control group(P<0.01),the anticoccidiosis index(ACI) was 163.1,The above results suggested that the rADF protein have Immunoprotective effect on E.tenella infection in chicken. |
PDF Full Text Request