Research Of Three Development-related Gene Of Eimeria Tenella

Coccidiosis is one of the most economically costly diseases of the poultry indu- stry, with estimated annual losses greater than three billion US dollars. Prophylactic feeding of coccidiostat drugs is the major disease control method used in commercial poutry. However, with increasing demands for high-protein meats and heightened consumer concerns over the use of antibiotics in poultry production, the search for alternative strategies against avian coccidiosis have intensied. Genetic engineering vaccine become more and more important for controlling the disease, and finding antigenic genes is the most difficultly work.1. Cloning and expression of BW4-C03,BW1-E03 geneIn order to understand the mechanism of invasion and development of Eimeria.tenella in our laboratory, we have gotten ESTs of differentially expressed genes of sporulated oocysts and sporozoites recerived by SSH and cDNA microarray .In this paper ,based on these ESTs, two novel genes including a complete open reading frame were obtained with RACE technique. BW4-C03,BW1-E03 were up-regulated in sporulated oocysts of E.tenella, Blast searches show that BW4-C03 sequence has more than 82% homologous both with the surface antigen 21- (EtmRNASAG21)and surface antigen23(EtmRNASAG23) of E.tenella, so the gene was named EtSAG. BW1-E03 sequence has 100% homologous with the surface antigen13(EtmRNASAG13). With bioinfomatics analysis software, property and function of proteins encoded by these novel genes were predicted, including physical and chemical property, trans-membrane structure, antigen site, conserved domain, function site. The two gene were detected by Real time PCR in four different stages (unsporulated oocysts,sporulated oocysts,sporozoites,,merozoites) and in different sporulated time(0 h,6 h,12 h,20 h,30 h,48 h). The result showed both of them were up-regulated in sporulated oocysts and have the highest copy number of transcription in 48h. A recombinant plasmids (pET-28C-EtSAG) was constructed successfully, The recombinant plasmids was transformed into BL21(DE3)for expression. After induction by IPTG, recombinant expressed proteins were identified by SDS-PAGE. The protein was expressed in the form of inclusion. Western-blot revealed that the protein has native antigens.2. Expression and preliminary founctional studies of ZB10-E05 geneIn this paper, an EST(cloning number is ZB10-E05) was chosed which is up-expressed in sporozoite, The full-length cDNA sequence was obtained by the rapid amplification of cDNA ends (RACE) technique. The protein encoded by ZB10-E05 has 46% homology with endonuclease/exonuclease of Toxoplasma gondii .A recombinant plasmids pGEX- 4T2-E05 were constructed successfully. the prokaryotic expression product was obtained. Western-blot analysis showed that the recombinant protein has good immunogenicity.The polyclonal antibodies was made by immunizing rabbits with the recombinant protein. In order to investigate the dispersed localision of the native proteins during the process of invasion and development of sporozoites. The sporozoites were purified from Eimeria. tenella sporulated oocysts and added into chicken fibroblast cell(DF-1), The process of invasion and development of sporozoites in DF-1 cells within 72h were observed by immunofluorescence microscopy. At the beginning, the target protein located at the top and middle of the worm;48h later it mainly located at the parasitophorous vacuole. The protein was up-expressed than other stages between 24h and 48h after invasion. In order to evaluate the implication of the protein in vasion of sporozoites of E.tenella , the labbled sporozoites were incubated with the pre-prepared purified anti-rabbit serum for 2h in different concentration(50 ug/mL,100 ug/mL,200 ug/mL,400 ug/mL). the sporozoites were added into DF-1 cell. The DF-1 cell was analyzed by flow cytometry instrument. The result showed: the inhibition rate had tendency to increase with the antibody concentration increasing,3. Evaluation of the efficacy of the recombinant protein E05For the purpose of evaluating the efficacy of proteins on induction of protection immunization against E.tenella challenge. Four groups of chickens(n=25) were immunized with two dose of E05(50μg,100μg),The chickens were challenged1×10~4 oocysts after 7 days of the second immunization .In this study, body weight gain,fecal oocyst shedding,intestinal lesion scores,humoral and cell-mediated immunity were studied . Chickens inmmuned with E05 were better than the control group when the chickens were attacked with sporulated oocysts, and 100μg purified recombinant protein was better than 50μg protein. it suggested that recombinant protein has partial protection against E.tenella changed. The level of antibody was detected by ELISA,The results showed antibodies was produced after chickens inmmued for 7 days and continued a long time.