大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

An Outer Membrane Protein IbeB Contributing toE.coli Crossing of the Blood-brain Barrier is Synthesized via a Precursor Molecule

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的

In order to further characterize the role of invasion locus ibe B in the pathogenesis of E.coli meningitis, the ibe B gene was subcloned into pET28a(+) and expressed in E.coli BL21 as recombinant proteins induced by IPTG.Two forms, a 50 kD precursor and a 34 kD mature protein, of IbeB proteins were obtained in this over expression system. Meanwhile, the data from in vitro translation experiments of the DNA fragments containing ibe B gene demonstrated that the synthesis of IbeB proceeded via a precursor protein. To analyze the cellular location of IbeB in E.coli , the protein was labeled with [ 35 S]methionine using the T7 expression system, and the 34 kD mature IbeB protein was identified as a component of the outer membrane proteins. More importantly, the antibody of recombinant 34 kD IbeB protein was able to block the invasion of brain microvascular endothelial cells (BMEC) by E.coli Kl in a dose dependent manner. These data suggested that the IbeB protein contributing to E.coli crossing of the blood brain barrier was an outer membrane protein synthesized via a precursor molecule. The results also indicated that forming of mature IbeB protein might be depending on intramolecular processing of IbeB precursor.