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目的研究霍乱弧菌(VC)O139群新类型ig1-rstR-ig2结构及功能。方法分别扩增CTXφ或nct-CTXφ基因组串联体的第一个基因组核心区末端和第二个基因组RS区起始端之间的基因,PCR产物测序及序列分析。扩增rstA的操纵区,克隆入pRS591质粒lacZ基因前,转化入JM109菌株,检测β-半乳糖苷酶活性,以反映rstA启动子强度。将不同类型rstR及启动子区分别克隆人pACYC184质粒,再将重组pACYC184质粒转化入携带不同重组pRS591质粒的JM109菌株中,检测β-半乳糖苷酶活性,以反映rstR表达产物RstR对rstA表达的影响。结果calc、ET和newO139类型ig1-rstR-ig2序列同源性分别为85.1%～91.6%、9.4%～17.7%和8.0%～26.2%。不同类型rstA的启动子强度不同,calc和ET类型的较高,newO139类型的较低。RstR对同类型rstA启动子活性表达均有明显的抑制作用,而对不同类型的则无明显抑制作用。结论阐明了霍乱弧菌(VC)O139群calc、ET和newO139类型ig1-rstR-ig2结构及功能。
Objective To study the structure and function of ig1-rstR-ig2, a new type of O139 cholera in Vibrio cholerae (VC). Methods The gene, PCR product sequencing and sequence analysis of the first genomic core region of the CTXφ or nct-CTXφ genome tandem body and the start of the second genomic RS region were amplified. Amplification rstA control region, cloned into the pRS591 plasmid lacZ gene, transformed into JM109 strain, beta-galactosidase activity was measured to reflect rstA promoter intensity. The different types of rstR and promoter regions were cloned into human pACYC184 plasmids, respectively, and the recombinant pACYC184 plasmids were transformed into JM109 strains carrying different recombinant pRS591 plasmids to detect the β-galactosidase activity to reflect the effect of rstR expression product RstR on rstA expression influences. Results The sequence identities ig1-rstR-ig2 of calc, ET and newO139 were 85.1% -91.6%, 9.4% -17.7% and 8.0% -26.2%, respectively. Different types of rstA promoter intensity, calc and ET types higher, newO139 type is lower. RstR on the same type rstA promoter activity were significantly inhibited, but for different types of no significant inhibitory effect. Conclusion The structures and functions of ig1-rstR-ig2 of calcite, ET and newO139 types of V. cholerae (VC) O139 group were clarified.