Cloning And Expression And Preliminary Application Of NcSRS2 Gene Of Neospora Caninum JL Strain

Neospora caninum,which is a protozoan parasite.N.caninum is obligate intracellular pathogen that appears to be capable of utilizing a fairly wide range of anumals as intermediate hosts.N.caninum can cause abortion and neonatal mortality in cattle and is now recognized to be responsible for significant economic and reproductive losses to be the livestock industry in certain geographical areas.Overseas scholar finded the surface protein NcSRS2 in N.caninum dense granule and tachyzoites.the surface protein NcSRS2 is prime candidates for mediating adhesion and host cell invasion.It is as to effective constituent of the ELISA,it may be suitable candidates for incorporation in N.caninum vaccines,the NcSRS2 gene encoding the surface protein of N.caninum is an important gene for the conservation of the genus.In this study,The NcSRS2 gene of N.caninum were cloned by polymerase chain reaction(PCR) using primers designed and the sequences were analyzed with molecular softpakage.The homogeneity of the NcSRS2 gene of N.caninum with the published gene(AY940488)is 99.8%,meaning the NcSRS2 gene fragment is the object gene.The NcSRS2 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing NcSRS2 gene were identified by restriction enzyme analysis and PCR method.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1 mmol/L IPTG in the form of inclusion bodies.The molecular weights of GST-NcSRS2 is 64.4 ku.The purity of the recombinant protein by affinity-purification of Glutathione Sepharose 4B was attained perfect result.Western-blotting analysis with positive antibodies against toxoplasma gondii showed the recombinant protein shared good antigencity and SpecificityThe antigenicity of the GST-NcSRS2 protein were identified using ELISA method.The results showed that the working concentration of the antigen is no lower than the 4μg/mL,the positive serum do 1:40 dilutions, the conjugate's optimal dilution was 1:2 000 instillations,the effective examine is the best.The method has no cross-reaction with Theileria sp, Toxoplasma tachyzoites and Babesia equi serm.The indirect ELISA and Xuan Xuenan's mathod parally were to detect N.caninum antibodies in 128 cattle serm samples.The results indicated that the disparation between two method was not pateney.The research can be used to detect N.caninum antibodies in cattle serm,rapidly,specificly and stably.