Nu Babesia Worms Bc-48 Gene Fragments In Escherichia Coli And Preliminary Application

Babesia caballi is a tick-borne protozoan parasite which causes fever, anemia, jaundice, and edema in horses and, in some cases, the death of the infected animals. This disease to be considered B disease by OIE, and leads to great economic losses in the horse industry. Rhoptry proteins were reported to be involved in the invasion of erythrocytes and might be suitable candidates for incorporation in vaccines against B. caballi merozoites, the BC48 gene encoding the rhoptry protein of B. caballi is an important gene for the conservation of the genus.In this study, The BC-48 gene of B. caballi were cloned by polymerase chain reaction (PCR) using primers designed and the sequences were analyzed with molecular softpakage. The homogeneity of the BC-48 gene of B.caballi with the published gene(U46551)is 97.1%, meaning the BC-48 gene fragment is the object gene.The BC-48 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing BC-48 gene were identified by restriction enzyme analysis and PCR mathod. 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- BC48 is 45 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 B. caballi showed the recombinant protein shared good antigencity and Specificity.The antigenicity of the GST-BC48 protein were identified using ELISA mathod.The results showed that the working concentration of the antigen is no lower than the 2.5μg/mL, the positive serum do 1:160 dilutions, the conjugate's optimal dilution was 1:4000 instillations, the effective examine is the best. The method has no cross-reaction with Neospora caninum,Toxoplasma tachyzoites and Babesia equi serm. The indirect ELISA and Xuan Xuenan's mathod parally were to detect B. caballi antibodies in 132 horse serm samples. The results indicated that the disparation between two method was not pateney.The research can be used to detect B. caballi antibodies in horse serm, rapidly, specificly and stably.