Surface display of lead-binding protein PbrR on Escherichia coli and preliminary study of intestinal ;colonization by the recombinant bacteria
10.3760/cma.j.issn.0254-5101.2016.12.010
- VernacularTitle:铅结合蛋白PbrR的大肠埃希菌外膜展示株构建及体内定植初步研究
- Author:
Changye HUI
;
Yan GUO
;
Chaoxian GAO
;
Xueqin YANG
;
Zhengyu LIU
;
Limei LI
;
Yuting CHEN
;
Wen ZHANG
;
Xianqing HUANG
- Keywords:
Lead-binding protein;
PbrR;
Bacterial surface display;
Colonization in vivo
- From:
Chinese Journal of Microbiology and Immunology
2016;36(12):935-941
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant Escherichia coli ( E. coli) with surface-dis-played lead specific binding protein PbrR and to further study intestinal colonization by the recombinant bac-teria in mice and gastrointestinal tolerance of the bacterial surface-displayed PbrR. Methods Chimeric pro-tein Lpp-OmpA coding sequence was chemically synthesized and inserted into the expression vector pET-21a to construct the outer membrane display vector pLOA. PbrR coding sequence was also obtained by chemical-ly synthesis and inserted into pLOA to generate the outer membrane display plasmid pLOA-pbrr. E. coli BL21 (DE3)pLysS was transformed with pLOA-pbrr and induced by IPTG. The expressed recombinant proteins were analyzed by 15% SDS-PAGE and Western blot assay. Lead adsorption capacity of the cell surface-dis-played PbrR in the simulated intestinal juice and tolerance of the recombinant E. coli to simulated gastric juice were analyzed, respectively. KM mice were orally given the induced recombinant bacteria by gastric lavage for 7 consecutive days and then were continually fed until day 30. The contents of recombinant bacte-ria in stool samples were detected by dilution plate method on day 7, 15 and 30. The recombinant protein with His tag was detected by immunoblotting on day 7 and 15. Results Based on Lpp-OmpA, the PbrR outer membrane display vector was successfully constructed. The recombinant fusion protein Lpp-OmpA-PbrR-His tag was highly expressed in E. coli. The recombinant E. coli strains displaying PbrR on their outer membrane accumulated a significant level of Pb2+ in simulated intestinal juice. Moreover, those strains showed a tolerance to gastric acid in vitro and could colonize in the intestinal tracts of mice via oral infection. The surface-displayed recombinant fusion protein showed a better tolerance to the environment of digestive tract. Conclusion The recombinant E. coli strain displaying PbrR on its surface showed a stronger capabili-ty of lead accumulation from simulated intestinal environment and could colonize in the intestinal tracts of mice. The surface-displayed recombinant PbrR also showed a good tolerance to digestive juice. This study paved the way for further researches on the selective elimination of lead by biosorption based on animal mod-els.
