Arabinose-controlled expression of htrA in Shigella flexneri through suicide plasmid-mediated homologous recombination
10.7644/j.issn.1674-9960.2015.05.008
- VernacularTitle:自杀质粒同源重组方法构建弗氏志贺菌htrA可控表达菌株
- Author:
Jing YANG
;
Lilan LU
;
Ping XU
;
Hongsheng LIU
;
Erling FENG
;
Hengliang WANG
;
Li ZHU
- Publication Type:Journal Article
- Keywords:
Shigella flexneri;
htrA gene;
suicide plasmid-mediated homologous recombination;
arabinose operon;
Western blotting
- From:
Military Medical Sciences
2015;(5):354-356,402
- CountryChina
- Language:Chinese
-
Abstract:
Objective To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.Methods Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector .The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting .Results The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot .Conclusion Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein .After being induced with arabinose , HtrA protein can be expressed normally .
