人HSP70表达载体的构建及在无细胞中的表达

Objective To construct the Expression Vector pCMV- HSP70 –myc and achieve the rapid expression of HSP70 in vitro cell-free expression system. Methods HSP70 gene open reading frame were cloned by PCR , which will be built into the pCMV-myc vector to form fusion expression plasmid after digestion. The protein would be expressed by rabbit reticulocyte in vitro cell-free protein expression system after the success of sequencing. Expressed protein were validated by Western blot . Results a pCMV-HSP70-myc vector was constructed Successfully , the molecular weight of expressed product of HSP70 in vitro cell-free system consistents with the theoretical according to Western blot technology, a rapid expression of HSP70 in vitro has been successed. Conclusions The pCMV-HSP70-myc fusion expression plasmid was successfully constructed and its effective fast in vitro cell-free expression system was also successful,which laid the foundation for exploring the biological function of HSP70 as well as the role of anti-tumor ,and provided a new platform for some transmembrane proteins difficult to express.