Huang S H, Wan Z S, Chen Y H, Jong A Y, Kim K S
Childrens Hospital Los Angeles and University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA.
J Infect Dis. 2001 Apr 1;183(7):1071-8. doi: 10.1086/319290. Epub 2001 Mar 8.
The ibeA gene (ibe10) previously identified by TnphoA mutagenesis is part of a 50-kDa full-length open-reading frame (ORF) encoded by a 1.37-kb DNA fragment. An isogenic in-frame deletion mutant of ibeA (ZD1) was constructed by chromosomal gene replacement with a suicide plasmid pCVD442 carrying a 2.1-kb DNA fragment with an ibeA deletion. Similar to the previously described TnphoA insertion mutant of ibeA, the isogenic ibeA deletion mutant ZD1 was significantly less invasive in human brain microvascular endothelial cells (BMECs) than the parent strain. The mutant ZD1 was fully complemented by the ibeA ORF. The ibeA gene was subcloned into pET28a(+) and was expressed as a recombinant protein with an N-terminal histidine tag. The recombinant IbeA protein had much greater activity (50 times) in blocking the invasion of BMECs by Escherichia coli K1 than did the partial protein fragment, which provides further evidence that ibeA is an important determinant for E. coli K1 invasion of BMECs.
先前通过TnphoA诱变鉴定的ibeA基因（ibe10）是由一个1.37 kb DNA片段编码的50 kDa全长开放阅读框（ORF）的一部分。通过用携带具有ibeA缺失的2.1 kb DNA片段的自杀质粒pCVD442进行染色体基因替换，构建了ibeA的同框缺失突变体（ZD1）。与先前描述的ibeA的TnphoA插入突变体相似，同基因的ibeA缺失突变体ZD1在人脑微血管内皮细胞（BMECs）中的侵袭性明显低于亲本菌株。突变体ZD1被ibeA ORF完全互补。将ibeA基因亚克隆到pET28a(+)中，并表达为带有N端组氨酸标签的重组蛋白。重组IbeA蛋白在阻断大肠杆菌K1对BMECs的侵袭方面比部分蛋白片段具有更高的活性（50倍），这进一步证明ibeA是大肠杆菌K1侵袭BMECs的重要决定因素。
