The Cloning And Prokaryotic Expression Of The SuAT1 Gene Of Bovine Theileria Annulata

Theileria annulata are tick-borne apicomplexans that multiplies in macrophage cell, leukocytes and in red blood cells. Theileriosis called tropical theileriosis, too, causing mild hyperthermia and anemia. The disease is epidemic widely in our country. The mortality reached 90%. Along with the development and tradement in cattle industry increased, the occurrence and epidemic frequency and epidemic area of theileriosis expands gradually in our country, theileriosis is one of the most economically important diseases of grazing cattle, which causes a debilitating disease of cattle and lead to major economic importance in China. The study showed that suAT1 had the potential to modulate the phenotype of infected cells. This process altered the infected cellular differentiation, proliferation, and apoptosis.The objective of this research was to study the immunogenicity and reactionogenicity of the suAT1 protein by cloning, expression and immunosorbent assay. According to suAT1 gene sequence of theileria annulata in Genbank, primers were designed and synthesized. The genomic DNA of theileria annulata was extracted. The suAT1 gene was amplified by polymerase chain reaction (PCR). The amplified fragment was inserted into pMD20-T vector. Then the recombinant vector were inserted into expressed vector pET-30a plasmid by two unique restriction sites of BamHI and HindⅢ. pET-30a-suAT1 was constructed. To identify pET-30a-suAT1, after PCR amplification and enzyme digestion and then the fragment was sequenced, then transformed into susceptible E.coli DH5α.After confirmation of the correctness of open reading frame of target gene in positive recombinant plasmid by sequencing, the gene fragment has a total length of 1377 bp, which encoded 459 amine acids. The homology of suAT1 gene sequence was 98.35% , the homology of the the Amino acid sequence was 97.13% comparing with a reported sequences. The results showed that suAT1gene had been cloned successfully.The recombinant plasmids pET-30a-suAT1 were transformed into the host strain of E.coli BL21(DE3)pLys and the target proteins were expressed by inducing with IPTG at 30℃. The expressed products of suAT1 gene were identified by SDS-PAGE. The results revealed that suAT1 gene had been expressed successfully. The products were 50.49kDa. The results showed that suAT1 protein were solubly expressed in bacteria, and suAT1 were expressed to form inclusion body. The expressed protein could be reacted with antibodies of theileria annulata by western blotting, which showed that the fusion protein have strong reactogenicity.This studies was the basis for studying the higher structure of the suAT1 protein, and provided structural basis for researching the interaction between the protein and the host cell DNA , which will be the basis of the target medicine .