猪水肿病毒素A亚单位基因的克隆及表达的尝试

Cloning and Expression of the A Subunit Gene of the Shiga like Toxin Type II Variant

以聚合酶链式反应 ( PCR)技术从大肠杆菌 TB1中扩增出猪水肿病毒素 ( SLT- IIe)的 A亚单位基因的 960 bp。将此 PCR扩增产物 ( slt- IIe A)在 Eco RI和 Bam HI位点间克隆进质粒p UC1 8中 ,获得了重组质粒 p1 8slt- IIe A,经 Micro Genic computer program分析证明在其整个序列中含有与已发表的 slt- IIe序列相同的序列 ,说明质粒 p1 8slt- IIe A是含有 slt- IIe A的重组质粒。切割重组质粒 p1 8slt- IIe A,将 slt- IIe A基因插入到表达性质粒载体 p GEX- 6p- 1中处于谷胱苷肽转移酶 ( GST)基因的下游 ,构建成重组质粒 ppslt- IIe A,并证明其中含有 slt- IIe A基因。然而 ,与原初预计结果相反 ,受到质粒 ppslt- IIe A转染的大肠杆菌 BL2 1 (名为重组质粒 PPSLT- IIe A)在不同温度条件、不同培养时间和不同 IPTG浓度下 ,却未表达预期的融合蛋白 GST- SLT- IIe A。本文对未能表达一事的可能原因进行了讨论。

The A subunit gene (slt IIeA,960bp) of the Shiga like toxin II variant (SLT IIe) was amplificated from the plasmid of Escherichia coli TB1 by polymerase chain reaction (PCR). The PCR product of slt IIeA was cloned into pUC18 plasmid between the EcoRI and BamHI sites,and the recombinant plasmid p18slt\|IIeA was obtained,which were analyzed with the MicroGenic computer program and showed that in its whole sequences contained a sequence in according with the published sequence of slt\|IIe.This demonstrated that the p18slt\|IIeA was the combinant plasmid containing the slt\|IIe.Cleaving the recombinant plasmid p18slt\|IIeA and cloning the slt\|IIeA gene insert into the expression plasmid vector pGEX\|6p\|1 downstream the gene coding for enzyme glutatione S\|transferace(GST) and the recombinant plasmid ppslt\|IIeA resulted,which was proved containing the gene slt\|IIeA.However,contrary to the original tentative anticipation,PPSLT\|IIeA,the recombinant E.coli BL21 tranfected with plasmid ppslt\|IIeA,did not expressed the interesting fusion protein GST\|SLT\|IIeA under the conditions of different culture tempretures,incubation times and IPTG concentrations.Factors accounting posibly for the fusion protein unablly expressed were discussed.