利用ctxb的自身启动子表达CTB

Secretive expression of cholera toxin B subunit by using its own promoter

霍乱毒素B亚单位 (CTB)在大肠杆菌表达体系中不能实现良好的分泌性表达。本文拟利用ctxb的自身启动子来实现CTB的高效分泌性表达。PCR方法扩增ctxb的调控序列和结构基因 ,克隆至pGEM T载体中 ,并在其下游链上大肠杆菌核糖体基因的转录终止信号rrnBT1T2 ,构建的表达质粒 pGEM T48转化大肠杆菌JM10 9和霍乱毒素基因阴性株霍乱弧菌IEM10 1。结果ctxb在自身启动子的调控下 ,大肠杆菌JM 10 9和霍乱弧菌IEM 10 1都实现了CTB的分泌性表达。但在 pGEM T48(IEM10 1)中CTB的分泌性表达量明显高于pGEM T48(JM10 9)中的量 ,两者比例为 5 0∶1。因此 ,pGEM T48(IEM10 1)

We attempt to express high cholera toxin B subunit by using its own promoter,which was found in cholera toxin A subunit structure gene,because the higher secretive expression of cholera toxin B subunit can not be done in E.coli.The ctxb regulatory sequence and ctxb structure gene were amplified by PCR,the PCR products were cloned into pGEM T vector.The expression plasmid designated pGEM T48 was constructed by connecting the transcription terminal sequence of rrnBT1T2 to the downstream of ctxb structure gene.The pGEM T48 was transformed separately into E.coli JM109 and V.cholerae IEM101 which is devoid genes encoding cholera toxin(CT) naturally.Although secretive expression of CTB was obtained in both host cells,the amount of CTB in IEM101 supernatant is higher than that in JM109,the ratio is 50∶1.Therefore,pGEM T48(IEM101) system is a highly efficient CTB secretive expression system,and the over expression of CTB can be used as adjuvant in peparation of vaccine against cholera infection.