Construction of Prokaryotic Expression Vector of Wheat TaCKX1 Gene and Its Expression

Objective: Prokaryotic expression vector of wheat TaCKX1 gene was constructed and was expressed to obtain 6×His·Tag fusion protein.Method: A pair of primers was designed according to digestion sites in plasmid pET-24a and the TaCKX1 gene sequence published by GenBank.The DNA fragment of 1 575bp was amplified by PCR from the pMD-QRCKX1 recombinant plasmid with TaCKX1 gene,then cloned into pET-24a and transformed into the host E.coli strain DH5α.The fragment was conformed to the original sequence.It indicated that fusion expression vector pET-TaCKX1 was constructed.The pET-TaCKX1 plasmid was transformed into BL21(DE3) plysS for expression.Induced by IPTG at 37℃,the expression product of TaCKX1 gene was identified by SDS-PAGE,and the expression condition was optimized.Result: TaCKX1 fusion protein had been expressed successfully in the form of inclusion bodies.The molecular weight is 58.9kD.Meanwhile,the condition of TaCKX1 fusion protein expression was induced with 0.5mmol/L IPTG for 15 hours.Wheat TaCKX1 gene was successfully expressed in E.coli,which was prepared for TaCKX1 polyclone antibody.Conclusion: Wheat TaCKX1 gene was successfully expressed in E.coli,which was prepared for TaCKX1 polyclone antibody.