重组质粒二级结构对丙型肝炎病毒E2蛋白基因表达的影响

Effect of gene structure of recombine plasmid in HCV/E2 protein Expression

将含HCV/E2基因的重组质粒 pUC18/HCV -E2的BamHI酶切基因片段和NcoⅠ +HindⅢ酶切基因片段分别克隆到原核表达载体pRSETHis中 ,构建两株重组HCV/E2重组表达质粒 pRSET·C/E2和 pRSET·A/E2。在诱导表达中 pRSET·A/E2的外源基因获得了高效表达 ,而 pRSET·C/E2的外源基因完全未得到表达。

HCV/E2 gene fragment was obtained from plasmid pUC18/HCV E2 by digesting with endonuclease BamHI,another E2 fragment was obtained from same pladmid by digesting with NcoⅠ and HindⅢ.Two fragments were inserted into proper reading frame of expression vector pRSETHis respectively,and recombiant expression plasmids of pRSET·C/E2 and pRSET·A/E2were constructed.these recombinant plasmids were transformed into E.coli.TOP10 strain and induced by IPTG.The expressive conditions were optimized,the induced purpose protein was detected and analysed by SDS PAGE and Western blot.The pRSET·A/E2 have a high level expression and the E2 fusion protein accounted for 20% in the total proteins of host bacteria meanwhile the pRSET·C/E2 have not detected expressive purpose protein entirely.We guess the reason of that is due to a very stable RNA secondary strcucture within mRNA translation initial region in pRSET·C/E2.