Studies On The Expression Of Major Suface Protein TNcSRS2Gene Of Neospora Caninum In E.Coli And P.Pastoris And Its Application

Neosporosis is a kind of global ectosarc disease caused by Neospora caninum. This disease can bring serious detriments to cattle. It is primarily causative agent of abortion, weaken fetus, stillbirth fetus, and sterility. The damage degree of this disease has obviously regional difference. Currently, there is no effective drug and vaccine for the control of neosporosis, and the only approach is eliminating the animal that infected with Neospora caninum. Therefor, the developments of a new detection kit and a subunit vaccine are particularly important.(Ⅰ) The recombinant plasmid pGEX-4T-tNcSRS2was transformed into host cell BL21, the target gene was successfully expressed in the form of inclusion body when induced with IPTG. The inclusion body of recombinant protein was purified with washing buffer. After being dialyzing, the recombinant protein was purified through glutathione agarose column. The protein concentration that determined by modified BCA protein assay kit was232.9μg/mL. An indirect rELISA kit for detection of antibody against Neospora caninum was developed using the purified protein as coating antigen. The developed kit was evaluated for specificity, sensitivity and reproducibility. There was no cross reaction with Toxoplasma gondii and Brucella. The result remained positive when the positive serum diluted to1600times. Both of the intra-assay and inter-assay variation coefficients were less than10%. The developed kit could be stable at least for6months at-20℃. The agreement between the developed kit and "IDEXX" Neospora caninum antibody test kit was90.45%, and93.46%with "Bio-X" Neospora caninum antibody test kit. Blood samples collected from1329animals in several suspected areas of Xinjiang Uyghur Autonomous Region were detected for antibodies against Neospora caninum by the kit, and the seroprevalence was11.8%. The detection results were verified by using commercial ELISA kits.(II) A pair of primers was designed for amplification of tNcSRS2gene. The tNcSRS2gene that was composed of990nucleotides was amplified by PCR from the genomic DNA extracted from the positive blood infected with N.caninum, then the tNcSRS2gene was cloned into pPIC9K vector to construct the recombinant expression vector pPIC9K-tNcSRS2. By using PCR, double digestion and nucleotide sequencing, identification, the result showed that the sequence homologous rates of nucleotide of tNcSRS2gene between this species and Nc-1was99.90%. After being linearized with Sal I enzyme digestion, the recombinant vector was transformed into Pichia pastoris by electroporation. The positive recombinants were selected using G418, then identification by using PCR. The recombinant yeast expressed recombinant protein with a molecular weight of37kDa and the optimal induction time was72h, the optimal methanol concentration was0.25%, the optimal pH value was7.0. Western bolt analysis showed that there was a specific band of37kDa and the recombinant protein was recognized by bovine anti-N.caninum serum. The NcSRS2protein of Neospora caninum was successfully expressed in Pichia pastoris, which lays the foundation for developing subunit vaccine by using the protein.