The effect on MexB expression with siRNA silencing mexB gene of Pseudomonas aeruginosa
10.3760/cma.j.issn.0254-5101.2011.11.001
- VernacularTitle:应用siRNA干扰技术沉默铜绿假单胞菌外排泵基因mexB的蛋白表达
- Author:
Ying SONG
;
Mingyou XING
;
Dong XU
;
Fengyun GONG
;
Chao XIA
;
Lili WANG
;
Xuhua XIE
;
Aixia SHEN
;
Weili ZHAN
;
Jianxin SONG
- Publication Type:Journal Article
- Keywords:
siRNA;
mexB gene;
Western blot;
Pseudomonas aeruginosa
- From:
Chinese Journal of Microbiology and Immunology
2011;31(11):961-966
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the alteration of MexB protein expression by plasmid containing siRNA template strand was transformed into Pseudomonas aeruginosa.Methods siRNA molecules specifically against mexB were designed and ligated into pGPU6/GFP/Neo vector to construct the recombinant plasmid pGPU6/GFP/Neo-siRNA.MexB gene was cloned into expression vector pET22b+ to construct plasmid pET22b+/mexB,the recombinant expression plasmid was transformed into E.coli BL21 (DE3)plysS and protein MexB was induced to express,then purified protein MexB was used to prepare specific antibodies in rabbits.The plasmids with siRNA molecules specifically against mexB were transformed into wild type strain,clinical multiresistant strain and mexB overexpression strain of Pseudomonas aeruginosa by electroporation respectively,and the changes of the expression of MexB were detected in 8,12,24 h respectively by Western blot.ResultspGPU6/GFP/Neo-siRNA was constructed successfully.Protein MexB was expressed successfully and the rabbit polyclonal antibodies against MexB was prepared well.The gene silence of mexB by siRNA molecules was effective in the three strains of Pseudomonas aeruginosa,but it depended on the silencing time.ConclusionThe expression of MexB was reduced in 8 h and 12 h,but in 24 h,the expression was unchanged.
