Smirnova N I, Krepostnova I M, Livanova L F, Zadnova S P, Eremin S A, Il'ina T S
Mol Gen Mikrobiol Virusol. 2007(4):7-13.
The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.
构建了接合重组质粒pIEM3（KmR TcR），以便将编码霍乱毒素B亚基的克隆ctxB基因导入霍乱弧菌细胞。该质粒是两个质粒共整合的结果：一个携带迷你卡那转座子和IS1元件的接合质粒pIEM1（KmR），以及携带克隆ctxB基因的pBR322衍生物pCTdelta27（TcR）质粒。选择无毒的霍乱弧菌埃尔托生物型菌株（根据PCR分析），其关键结构和调控致病性基因缺失，且O1抗原的单个基因发生突变，作为pIEM3质粒的载体菌株。结果表明，在5%的霍乱弧菌细胞中发生了共整合解偶联，随后仅保留了多拷贝的pCTdelta7质粒。这一事件导致形成了TcRKmS克隆，其特征是霍乱毒素分泌的B亚基产量高（10至14微克/毫升），其中一个（KM93）被选为该蛋白的生产菌株。采用分子遗传学和生化分析方法来阐明克隆的ctxAB基因在KM93细胞内的遗传和表达特性。结果表明，克隆的ctxB基因的表达与toxR、tcpP、tcpH、toxT调控基因无关，这表明可能存在其他机制对霍乱毒素B亚基基因的转录活性进行调控。如果在工业生产条件下培养构建的生产菌株，也会观察到霍乱毒素B亚基的有效产生。这表明它有可能作为参与制造霍乱免疫诊断和预防制剂的这种蛋白质的来源。
