Prokaryotic Expression Of P33 Major Surface Protein From Theileria. Sergenti

Theileriosis is one of blood protozoal disease, caused by Theileriafamily, Theileria genus, Theileris sergenti. It multiplies in cattleintererythrocytes. which results in serious economic losses in animalhusbandry. Recent years, Theileriosis occurre increasing tendency, bothdisease rate and fatality rate are high in epidemic area, which causedaboard importance of world. T. sergenti P33 protein is one of T. sergentimajor surface protein, and has strong immunogenicity. There ishydrophobic membrane anchor on the C end, and signal peptide on the Nend of P33 gene sequence.A pair of syntheticoligonucleotide primers which designed accordingto the gene encoding P33 major surface protein of Theileria sergenti, were used to amplify the parasite DNA from the blood ofT. sergenti-infected cattle. Homology of nucleotide sequence between thecloned P33 gene and the published sequence. from GenBank was 99ï¼…, whichproved the purpose gene fragment was P33 major surface protein ofTheileria sergenti. P33 major surface protein gene was ligated intopET-28a(+) vector and was certified by PCR amplification and digestionwith endonuclease., which proved a prokaryotic expression vector that.containing the pET-28a-P33 was constructed successfully. The expressedP33 protein, molecure 30.3 ku, and it was 43ï¼…of the total bacteriumprotein, lt exists. in cytorrhyctes status. The constructed pET-28a-P33vector was induced by lm mol/L IPTG and expressed highly in EscherichiacoliRosetta (DE3), it reached the highest point at the 4 hour. then theexpressed protein was analyzed by Western-blotting. Result showed that the protein can recognize positive serum of Theileria sergenti, and ithas strong reactivity.