Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12.
Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12.
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大肠杆菌 K-12 中霍乱弧菌肠毒素基因的分子克隆。
DOI：
10.1073/pnas.79.9.2976
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发表时间：
1982
影响因子：
11.1
通讯作者：
Mekalanos,JJ
中科院分区：
文献类型：
--
作者：
Pearson,GD;Mekalanos,JJ
利用大肠杆菌热不稳定肠毒素（LT） A亚基和B亚基基因的杂交探针，对霍乱弧菌569B株的DNA进行了霍乱毒素基因序列分析。Southern blot分析表明，霍乱毒素A和B亚基基因在菌株中均有重复。在质粒pBR322中克隆到一个5.1千碱基的毒素亚基基因对。携带重组质粒pJM17的大肠杆菌细胞显示产生霍乱毒素，发现霍乱毒素主要与细胞相关。蛋白质化学分析表明，该毒素以其非缺口形式存在，需要胰蛋白酶进行额外的蛋白水解处理才能在组织培养中表现出充分的毒性。大肠杆菌中霍乱毒素分泌和蛋白水解过程的改变与之前在lt中观察到的相似。pJM17上的A亚基基因在体外产生的插入突变被证明可以消除A链的产生，但仍然允许B链的产生。这些观察结果以及限制性基因图谱数据表明，霍乱毒素和LT基因在遗传组织上非常相似。
Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.