摘要
目的构建副溶血弧菌calR的基因突变株和回补株,为研究CalR的功能奠定基础。方法 PCR扩增calR基因的同源臂融合片段,并直接克隆入自杀质粒p DS132中。通过接合转移的方式将重组自杀质粒转入副溶血弧菌RIMD2210633株(WT)中,利用同源重组的方法替换原始calR基因以构建calR无痕突变株(ΔcalR)。PCR扩增calR的基因序列,并将其直接克隆入p BAD33质粒中,构建回补质粒。将回补质粒转入到ΔcalR中,即得回补株(ΔcalR/calR∷p BAD33)。将vop N的启动子区克隆入p HRP309质粒的β-半乳糖苷酶基因上游,构建Lac Z重组质粒,并将该重组质粒分别转入WT/p BAD33、ΔcalR/p BAD33和ΔcalR/calR∷p BAD33中,通过测定并比较3株菌中β-半乳糖苷酶活性的差异来判定CalR对vop N的调控关系。结果与结论 Lac Z结果表明,CalR负调控vop N的转录,且回补株的回补效果良好,表明成功构建了副溶血弧菌calR基因的突变株和回补株,为后续对CalR的功能研究打下了基础。
Objective To construct the calR null mutant and the complemented mutant strains in Vibrio parahaemolyticus（ VP）. Methods The homologous flanking fragments of calR were amplified by using fusion PCR,and then cloned into suicide plasmid p DS132. The recombinant suicide vector was transferred into VP RIMD2210633（ WT） by conjugation. The calR null mutant（ ΔcalR） was screened and verified by PCR. The calR coding region was amplified,purified and cloned into the p BAD33 vector harboring an arabinose p BAD promoter. Then,the recombinant plasmid calR∷p BAD33 was introduced into ΔcalR through electrotransformation,yielding the complemented mutant strain ΔcalR / calR∷p BAD33. The promoter-proximal region of vop N was cloned into the p HRP309 vector containing a promoterless lac Z gene. The recombinant Lac Z reporter plasmid was transformed into WT / p BAD33,ΔcalR / p BAD33 and ΔcalR / calR ∷ p BAD33,respectively,to measure the promoter activity（ the β-galactosidase activity） of the target gene（ vop N） in these three strains by using the β-galactosidase enzyme assay system. Results and Conclusion The Lac Z fusion assay indicated that the promoter activity of vop N was enhanced in ΔcalR / p BAD33 relative to WT / p BAD33 that was comparable to ΔcalR / calR∷p BAD33,suggesting the negative regulation of vop N by CalR and simultaneously the successful construction of calR null mutant and the complemented mutant strains in VP.
出处
《军事医学》
CAS
CSCD
北大核心
2016年第5期374-378,共5页
Military Medical Sciences
基金
江苏大学高级人才科研启动基金项目(14JDG166)
病原微生物生物安全国家重点实验室开放研究基金资助项目(SKLPBS1438
SKLPBS1437)
关键词
副溶血弧菌
calR
突变株
回补株
Vibrio parahaemolyticus
calR
mutant strain
complemented mutant strain
