Location And Immunoprotection Of Rhomboid Protein For E.tenella

Coccidiosis which is caused by several species of the genus Eimeria is an important disease which harmed severly to modem chick-keeping and exists in widely. E.tenella is an important specie among those protozoans. Until now many problems need to be solved such as drug-resistance and so on. Signal transduction of EGFR and EGF is a hot spot and ahead field in nowadays.The Rhomboid,EGFR and EGF are related the adhesion and invasion of E.tenella to host cells had been found.The Rhomboid gene of E.tenella is a new gene. In the experiment, prokaryotic expression vectors of Rhomboid was constructed and expressed in E.coli.The vaccine could induce protective immune responses against E.tenella after chicken were inoculated.Prokaryotic expression and clone of the Rhomboid gene The fragment of Rhomboid ORF was amplified by PCR with designed primers according to Rhomboid. The recombinant plasmids pMD18-T-Rhomboid was constructed. The ligation products were transformed to competent cells of host strain DH5a. The recombinant plasmids pET-28a-Rhomboid was constructed and transformed into E.coli Rosetta (DE3) , and the cells were cultured and induced by IPTG. The expressive levels were determined by SDS-PAGE and Western blot analysis of the cell lysates. The localization of Rhomboid in E.tenella was determined by irnmunofluorescence using antibodies specific for rRhomboid. The results showed that the recombinant proteins were highly expressed induced by 1mM IPTG for 4h before harvesting cells. The yields of rRhomboid were up to 60% of the total bacterial protein in the cell lysates. The recombinant proteins were inclusion bodies in the cytoplasm and were specificity of E.tenella by Western blotting.Antigen location Prepared succeedly recombinate proteantigen of the E.tenella with extraction and renaturation purification of the cytoryctes.Gained ideal result with immunizing BALB/c mouse or coating antigen and three hybridomas of stable secreting Rhomboid-McAb were selected.The subclasses of McAb was IgG₁. hnmunofluorescence staining of sporozoites showed that Rhomboid protein was present on the surface of sporozoites. It may be the vaccine candidate antigen excellently.Immunoprotection response induced by rRhomboid protein against E.tenella in chiken. The responses of specific humoral and cellimmunity were detected by indirect ELISA and flow cytometry after the chiken were immunized with rRhomboid protein by different injection. The immunoprotection of rRhomboid protein against E.tenella was studied after the chiken were inoculated with E.tenella oocysts. The results showed that the responses of specific humoral and cell immunity in chiken were elevated by inoculating rRhomboid protein. Oocysts number of immunized mice were decreased significantly compared with control groups (P< 0.05). The highest rate of immunoprotection of immuned group is 36.3%.. The above results suggested that the rRhomboid protein have Immunoprotective effect on E.tenella infection in chiken.