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目的考察Pbr R大肠埃希菌外膜展示株在不同条件下的体内外生长特性,为其体内定植驱铅能力的研究奠定基础。方法 15%SDS-PAGE分析不同浓度诱导剂L-阿拉伯糖诱导展示菌p BAD-loa-pbrr/DH10B的重组蛋白表达情况;铅平板敏感实验对比重组菌诱导及非诱导条件下在含铅平板上的菌落形成能力;体外培养实验考察诱导剂的加入对重组菌生长能力及耐铅能力的影响;通过混合共培养实验对比诱导条件下重组菌与空宿主菌的竞争生长能力;考察重组菌6 h内的胃酸耐受性;考察诱导剂及染菌方式对重组菌小鼠体内定植的影响。结果低至0.002%的L-阿拉伯糖即可诱导重组蛋白的高效表达;Pbr R展示菌对铅表现出了一定的耐受力;诱导剂引入后重组菌生长能力降低;重组菌对胃酸有较好的抗性,经口染菌方式重组菌可定植于小鼠肠道,但摄入L-阿拉伯糖水后,粪便中重组菌快速丢失,通过连续染菌方式可维持其定植水平。结论重组菌体内外培养实验结果表明,基于商品化的诱导型表达载体构建的展示菌难以表达重组蛋白的同时实现小鼠体内的稳定定植。
Objective To investigate the in vitro and in vivo growth characteristics of Pbr R Escherichia coli outer membrane display strains in vitro and in vivo, and to lay a foundation for the study on the ability of Pbr R to screen lead in vivo. Methods The expression of recombinant protein of pBAD-loa-pbrr / DH10B induced by different concentrations of L-arabinose was analyzed by SDS-PAGE. The sensitivity of lead plate was compared with that of lead-free plate In vitro culture experiment to investigate the effect of inducing agent on growth ability and lead-tolerant ability of recombinant bacteria; compare the competitive growth ability of recombinant bacteria and empty host bacteria under the induction conditions by mixed co-culture experiment; Gastric acid tolerance; investigate the effect of inducing agents and bacteria on the colonization of mice in vivo. As a result, the expression of recombinant protein was induced with L-arabinose as low as 0.002%; Pbr R exhibited some resistance to lead; the growth ability of recombinant bacteria decreased after the introduction of inducer; Good resistance, oral infection by recombinant bacteria can be colonized in the intestine of mice, but the intake of L-arabinose water, the rapid loss of recombinant bacteria in the stool, through continuous bacterial contamination can maintain its level of colonization. Conclusion The results of in vitro and in vivo culture of recombinant bacteria showed that the display bacteria constructed on the basis of commercial inducible expression vector could not express recombinant protein and achieve stable colonization in mice at the same time.