Prokaryotic Expression, Identification Of Human Cytomegalovirus Pp65 Fragment And Establishment Of Its Eukarvotic PPIC9K Expression Vector

Objective To select the pp65 gene fragment containing the predominance epitopes and clone the pp65 gene fragment.Then,to construct the prokaryotic expression system for expressing the pp65 fragment and identify the immunological character of the expressed protein.And also to establish the pPIC9K eukaryotic expression vector.Methods pp65 gene was amplified by PCR.The primers were designed with the multi-clone sites in the expression plasmid pET21a(+)and the pp65 gene.The temple was pGEM-T-pp65 plasmid containing the total pp65 gene.Through BamH I and Xho I double digesting,T4 ligase ligating,blue-white spot screening,the pp65 fragment was subcloned into the pET21a(+) expression vector so called pET-21a(+)-pp65.The identified pET-21a(+)-pp65 was transfected into E.coli BL21(DE3).The protein was purified by His-tag nickel post and identified by western blotting and ELISA.The pp65 gene was amplified by PCR.The primers were designed with the multi-clone sites in the expression plasmid pPIC9K and the pp65 gene.The pp65 fragment was ligated with the pMD19-simple.The exactly identified pMD19-simple -pp65 was digested with SnaBI and BlnI,then ligated with the digested pPIC9K.The pp65 fragment was subcloned into the pPIC9K expression vector so called pPIC9K -pp65.Then pPIC9K -pp65 was transfected into DH5α.Results In this research,HCMVpp65(1087—1515nt)was cloned.The reconstructed plasmid pET-21a(+)-pp65 was constructed successfulIy.Prokaryotic express system have been acquired.The fused pp65 protein had activity by identified with western blotting and ELISA.In addition,pPIC9K-pp65 was constructed successfully as well.Conclusions Prokaryotic express system have been acquired and the active and purified pp65 protein was acquired.In addition,pPIC9K-pp65 was constructed successfully.All these support the research of sub-unit vaccine and detecting kit.It is the foundation of expressing active protein and as the elementary experiment for biologic function of pp65.