赖型钩端螺旋体017株外膜蛋白新基因ompL17基因靶向敲除及其突变株的构建

Construction of suicide vector of a novel gene ompL17 encoding the outer membrane protein of Leptospira serovar lai strain 017

目的将外膜蛋白新基因ompL17靶向敲除并构建该基因的突变株。方法提取钩端螺旋体017株基因组DNA,扩增出外膜蛋白新基因ompL17,与打靶载体p2NIL重组,并插入氨苄抗生素抗性基因Ampr+使外膜蛋白新基因ompL17失活,构建重组的基因打靶质粒p2NIL17A。电穿孔转化入钩体017株中构建突变株,通过豚鼠模型观察该突变株的毒力变化。结果PCR、Dotblot和酶切分析,证实构建了以氨苄青霉素抗性基因DNA为标记探针的基因打靶载体,并构建外膜蛋白新基因ompL17敲除的钩体017株突变株。结论成功将钩体外膜蛋白的新基因ompL17进行靶向敲除,并构建钩体017株突变株,为进一步进行该基因的功能研究和阐明赖型钩体致病的分子机制奠定了基础。

Objective To knock out Leptospira interrogans ompL17 gene encoding an outer membrane protein and construct the mutant of ompL17 gene. Methods The novel gene ompL17 was PCR-amplified from the genome DNA template of Leptospira interrogans strain 017. The cloned gene was ligated to plasmid p2NIL and antiAmp gene. The recombinant p2NIL17A plasmid was transformed into Leptospira strain 017 by electroporation method. The virulence change was determined through cavy-medel. Results PCR, Dot blot and restriction endonuclease map revealed the successful construction of the gene target vector and the mutant strain 017 by ompL17 gene knock out. Conclusion The result of this study lays foundation for further exploring biological functions of the ompL17 gene and molecule pathogenesis of Leptospira interogans serovar Ira.