The Expression Of P33 Gene Of The Theileria Sergenti In The Yeast

Theileria sergenti is part of Taylor worm family and genus,it is a kind of blood parasite that parasitic in animals,mainly live on the cow’s lymphocyte,red blood cells,macrophage sand spread by ticks.At the present,cattle theileriosis sergenti present a tendency of increasing frequency in recent years with the continuous development of cattle and harm the cattle industry.There are no specific medicine and vaccine to treatment and prevention the disease so far.The p33 gene is the main surface of the protein gene in the boby and has good sex immunogenicity and reactions,thought to be the ideal candidate antigens,Eukaryotic expression system,besides has the advantages of the prokaryotic expression system,but also has high express foreign protein content,stable genetic traits,purification of the product is simple,and many other advantages,attract more and more people.The experimental results show that.Eukaryotic expression system,in addition to the advantages of prokaryotic expre ssion system,but also has a high expression of foreign protein,genetic traits stable,si mple product purification and many other advantages,by more and more people favor.In this study,according to Genbank T.sergenti p33 gene sequence of Chinese plants,design a pair of specific primers,using conventional PCR amplification the p33 purpose genesize that has size of 786 bp,build the pMD-19-T-p33 recombinant plasmid through to clone the pMD-19-T carrier then sequencing analysis by PCR and double enzyme identification,confirmed the purpose fragment cloned correctly to the pMD-19-T carrier.The p33 protein gene was cloned to expression vector pPICZ a A of yeast A recombinant plasmid by PCR,EcoR I and Xba I double enzyme identification,proved successful construction of eukaryotic expression vector containing the purpose gene pPICZ α A-p33.Then convert it into GS115 yeast cells and received high copy restructuring through the 800 μg/ml concentration of bleomycin filter finally then after screening positive strains through PCR,used the methanol of 1%To induced and strongly expressed of foreign proteins in pichia in the pichia then gained protein that size of 29.67 Ku by SDS-PAGE to the induction of production,onsistent with expected results.Then the induced proteins was authenticated of Western blot reactogenicity,protein can be specificity recognized by positive blood-serum of Theileria.sergenti of bull and showed that the protein has a good response to the original.The result of experiment had lay a solid foundation to diagnostic kit.Of disease of theileria sergenti and p33 gene of theileria sergenti of monoclonal antibody and the study of vaccine research of theileria sergenti.