弗氏志贺菌M90T株毒力相关膜蛋白复合物组成基因缺失突变体的构建  

Construction of Gene Knock-Out Mutants of Components of a Virulence Related Membrane Protein Complex of Shigella flexneri M90T

作　　者：冉金宝[1,2] 廖翔[2] 周围[2] 王羽[1,2] 魏波[1,2] 高原[2] 岳俊杰[2] 梁龙[2] 王纯洁[1] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2012年第6期777-780,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30970122)

摘　　要：目的:构建弗氏志贺菌M90T株毒力相关膜蛋白复合物组成蛋白基因的缺失突变株。方法:分别扩增目的基因的上、下游同源臂和卡那霉素抗性基因片段,利用重叠延伸PCR技术将这3个片段融合为打靶片段,电击转化M90T/pKD46感受态细胞,在λRed重组系统的作用下,通过同源重组将目的基因置换为卡那霉素抗性基因,之后在辅助质粒pCP20的作用下切除FRT位点之间的卡那霉素抗性基因,得到基因缺失突变体。结果与结论:分别构建了M90T株毒力相关膜蛋白复合物4个组成蛋白Apy、DnaK、ClpB、YdgA的基因缺失突变体,为进一步研究各基因的功能提供了突变体。Objective： To knock out components of related membrane protein complex of Shigella flexneri M90T coding gene respectively and obtain the corresponding mutant. Methods： DNA fragments composed of flank se- quences of target gene and kanamycin resistance gene between them were obtained by overlap extension PCR, and then transferred into M90T/pKD46 to replace target gene with kanamycin resistance gene by homologous recombina- tion with help of kRed recombination system. The kanamycin resistance gene between FRT sites was subsequently removed with help of plasmid pCP20. Results ＆ Conclusion： Four gene knock-out mutants of components Apy, DnaK, ClpB and YdgA of a virulence related membrane protein complex of M90T were successfully constructed. These mutants will be applied in the function studies of these genes.

关 键 词：膜蛋白复合物 缺失突变株 重叠延伸PCR λRed重组系统 弗氏志贺菌M90T株 

分 类 号：Q78[生物学—分子生物学]