Established The Eimeria Tenella Model In Vitro With Cloning And Expressing The Gene Of HSP70

Chicken coccidiosis is the parasitic protozoal disease caused by one of seven kinds of Eimeria alone or mixed in different parts of intestine.(1)In this study,using chicken intestinal epithelial cells (IEC), instead of renal cell, a new type of chicken Eimeria culture model was established in vitro, because the condition and process of IEC infected with Eimeria is closer to that of the natural infection of chickens. The results of isolation and culture of chicken IEC in vitro showed that the chicken IEC adhered to the wall of plate after 24-48 hours, and formed into monolayer in 7～8 d, when inoculated to culture plate after digestion with typeⅠcollagenase 70 min in 37℃and low-speed centrifugation, and under the culture conditions of 5% CO2, 39.5℃, DMEM containing 5% fetal bovine serum.(2)The purified sporozoites were collected, and inoculated to the primary embryo IEC. Using the culture model, the reproductive conditions of E.tenella schizony stage were explored, and the optimal medium and added components for E.tenella culture were determined, and the adaptive E.tenella was established in the chicken IEC. The results showed that sporozoites invaded into IEC in 2 hours when sporozoites were inoculated to the IEC cultured 8 days later. The medium was changed again, and the results weree observed once in every 12 hours. The first generation of schizonts were observed in 3-4 days, the second generation of schizonts were observed in 5-7 days, and the oocysts were appeared in 7-8 days post-inoculstion. The culture model in vitro laid new ways for the researches in the physiochemistry, immunology, vaccine preparation, medical effecacy, and the prevention and treatment of chicken coccidiosis.(3)The specific primers were designed according to HSP70 gene, the gene sequence of E.tenella Guangdong strain (GenBank Locus: Z46965.1) was amplified, then the gene was cloned into expression vector, and transformed into E.coli Rosetta. The expression of protein in host bacteria was achieved with IPTG induction. The HSP70 protein expressed with vector PET-32a (+) was in the form of inclusion in the bacterial cells. The soluble fusion protein was expressed with vector pMAL-c2X after repeated determination of expression conditions. The higher concentration of protein was obtained by Amylose Resin chromatography. The HSP70 gene was inserted into vector pcDNA-6 to construct the DNA vaccine expression vector. The pcDNA-6-HSP70 plasmid was transfected into Vero cells. The results showed that HSP70 protein was successfully expressed in eukaryotic cells.(4)The seral antibody titers of E. tenella infected chicken immunized with recombinant proteins were detected by ELISA. The parameters including cecal lesion score, oocysts per gram feces (OPG), weight gains, and anti-coccidial index (ACI) were observed. The results showed that the recombinant proteins induced certain degree antibody levels and anticoccidial immunoprotection.