霍乱弧菌O139群新类型ig1-rstR-ig2结构和功能研究

Study on structures and functions of new types of ig1-rstR-ig2 of Vibrio cholerae O139 serogroup

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 investigate he structures and functions of new types of ig1-rstR-ig2 of Vibrio cholerae （VC） 0139 seregroup. Methods Genes including end of core region and start of RS region of CTXφ or nct-CTXφ genome were obtained with PCR method. Operator of rstA was obtained with PCR method and cloned into the front of lacZ gene of plasmid pRS591, and then transformed into JM109 strain. β-galactosidase of recombinant JM109 was detected quantitatively for the activity of each rstA promoter. Different type of rstR gene and promoter region was cloned into plasmid pACYC184 differentially, and each recombinant plasmid pACYC184 was transformed into recombinant JM109 strain including different recombinant plasmid pRS591 differentially. β-galactosidase of each JM109 strain including two recombinant plasmids was detected quantitatively for observation of regulating function of each type of RstR on expression of each type of rstA gene. Results The homology of calc, ET and newO139 types of ig1, rstR and ig2 were 85.1%-91.6%, 9.4%-17.7% and 8.0%- 26.2%. Activities of promoters of rstA were different, those of calc and ET types were higher,that of new O139 type was lower. Repression of expression of rstA by the same type of RstR was prominent, but was not prominent in other types. Conclusions Structures and functions of calc, ET and newO139 types of ig1-rstR-ig2 of O139 serogroup Vibrio cholerae were elucidated.