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

Surface display of lead-binding protein PbrR on Escherichia coli and preliminary study of intestinal colonization by the recombinant bacteria

目的 构建铅特异性结合蛋白PbrR的大肠埃希菌外膜展示株，并考察其在小鼠体内的定植及外膜展示的重组蛋白在消化道内环境耐受能力。方法 全基因合成嵌合蛋白Lpp-OmpA编码序列，将其插入表达载体pET-21a，构建外膜蛋白展示载体pLOA。全基因合成PbrR编码序列，将其插入pLOA，构建PbrR外膜展示质粒pLOA-pbrr。转化表达宿主大肠埃希菌BL21（DE3）pLysS，IPTG诱导表达，15% SDS-PAGE及Western blot分析表达情况。考察PbrR外膜展示株在人工肠液中的铅吸附能力及在人工胃液中的存活能力。KM小鼠经口连续7 d给予诱导后的展示株，终止染菌后，继续饲喂至30 d，稀释涂平板法检测第7、15、30天小鼠粪便中重组菌含量，免疫印迹法检测第7和第15天小鼠粪便中的重组蛋白残留。结果 基于Lpp-OmpA展示载体，成功构建了PbrR的外膜展示质粒。融合蛋白Lpp-OmpA-PbrR-His tag得到了高水平表达，展示株在人工肠液中表现出了显著升高的铅离子吸附能力。展示株表现出了一定的胃酸耐受性，经口染菌后可定植于小鼠肠道，外膜展示的重组融合蛋白对消化道内环境表现出较好的抵抗能力。结论 大肠埃希菌PbrR外膜展示株在体外模拟肠道环境中表现出了较强的铅富集能力，体内可定植小鼠消化道，外膜展示蛋白也有较好的消化液耐受能力。本文为进一步基于动物模型的生物吸附选择性驱铅的研究提供基础资料。

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 bacteria in mice and gastrointestinal tolerance of the bacterial surface-displayed PbrR. Methods Chimeric protein 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 chemically 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-displayed 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 bacteria 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. coll. The recombinant E. coli strains displaying PbrR on their outer membrane accumulated a significant level of Pb^2＋ 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 capability 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 models.