Cloning and expression of brown planthopper carboxylesterase in Escherichia coli
Wu Yan · 2006
The purpose of the study is to clone,identify and express the mature protein gene of carboxylesterase from brown planthopper(BPH) and to play a foundation for the study of carboxylesterase′s characters and for the clarification of the role that carboxylesterase plays in the process of the transformation of BPH′s virulence.The gene encoding carboxylesterase was amplified from cDNA of BPH by using PCR technique,and the PCR product was about 1 900 bp long.Clone vector pMD18-T-cae was successfully constructed by using T-A clone technique.The gene(cae-M) encoding mature protein of carboxylesterase was amplified from the plasmid pMD18-T-cae,and the PCR product was about 1 600 bp long.The PCR product of cae-M and pET28a were digested by EcoRⅠ and HindⅢ double enzymes.The prokaryotic expression vector pET28a-caeM was constructed by using the purified cae-M that was cloned into the expression vector pET28a.The plasmid containing pET28a-cae-M was transformed into competent E.coli BL21(DE3).The bacteria were induced by IPTG and their lysates were loaded onto SDS-PAGE.An approximately 62 kD protein was observed on the SDS-PAGE.The protein was analyzed by using Western-blotting and it has antigenic activity of His-tag.