Cloning And Expression Of The SRS2 Gene In The Neospora Caninum And Establishment Of ELISA For Neosporosis

Neospora caninum is one of intracellular protozoan parasite which is responsibe for neuromuscular disease and abortion in cattle, dogs and other animals,especially, abortion of cow cattle, which has brought about enormous economic loss for the livestock farming. Early diagnosis is important for controlling neosporosis. It has been shown that NcSRS2 , the major surface antigen of Neospora caninum is a highly immunogenic protein and an appropriate antigen for development into a caccine. Therefore, NcSRS2 was chosen as the surface protein for development of indirect ELISA in neosporosis for diagnosis in cattle .According to NcSRS2 gene sequence of Neospora caninum, two pairs of primers were designed and synthesized, which were used to amplify the fragments of NcSRS2 gene with PCR in the 163～1141, and then the corresponding T/A clones were constructed with the part fragments. PCR amplification and enzyme digestion after identifying the fragment, then the fragment was sequenced and subcloned into pET-32a plasmid, then transformed into susceptible E.coli DH5α. The positive clones were selectec from the ampicillin positive LB plate after identification by PCR and enzyme digestion. The expression of the target genes was induced with 1mmol/L IPTG after all the recombinant plasmids were identified and transformed into the competent cells of the host cell E.coli BL21. The fusion protein NcSRS2t was expressed. The expressed protein could be reacted with antibodies of N.caninum by western blotting.The recombinant SRS2t was purifyed and thereafter evaluated in an enzyme-linked immunosorbent assay(ELISA) for serological diagnosis of Neosporasis. The positive and negative of Neospora caninum sera were used for developing an indirect ELISA for SRS2 antibody detection. The suitable concentration of antigen was determined by square matrix titration,and the specificity and repetitiveness of this method were determined. 96-well plates were coated with 5μg/mL commercially synthesized SRS2t protein, optimal blocked with 1% gelatin at 37℃for 2h, optimal serum samples were diluted by 1︰200 and incubated at 37℃for 1h, optimal HRP-conjugated secondary antibodies were diluted by 1︰5000 and incubated at 37℃for 1h, and the chromogen, TMB was added and after 15min incubation at 37℃, the reaction was stopped by 2mol/L H2SO4. The cut-off OD of positive and negative is 0.275.