炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体的构建

Construction of Putative S-Layer Protein SLP Deletion Mutant in Bacillus anthracis

目的:构建炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体,以进行后续SLP的功能研究,为炭疽芽孢杆菌重要基因功能的研究建立技术平台。方法:利用PCR技术,分别扩增得到目的基因的上游同源臂(slp-F)和下游同源臂(slp-R),将抗性基因(S)和上、下游同源臂先后连入穿梭质粒pKSV7,构建打靶载体pKSV7-FSR,经去甲基化后,电转化入炭疽芽胞杆菌A16R,通过同源重组敲除slp基因,并通过DNA测序和Western blot实验验证;对野生株和突变株37℃时的生长曲线及生化反应进行比较研究。结果:分别从DNA水平和蛋白质水平证实slp基因被成功敲除;突变株对数期生长较快,衰退较慢,与野生株的生化反应差异不明显。结论:获得了炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体。

Objective： To construct a putative S-layer protein SLP deletion mutant of Bacillus anthracis for exploring the function of putative S-layer protein in Bacilius anthracis and providing the basis for further study of important genes of B. anthracis. Methods： To construct targeting vector pKSV7-FSR, the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of slp were cloned into the plasmid pKSV7 in tandem. After demethylation of targeting vector, the recombinant vector was introduced into B.anthracis A16R by electroporation. The slp gene was deleted through homologous recombination and A16R△slp was confirmed by DNA sequencing and Western blot. Growth of the mutant and wild strain at 37℃ were measured respectively, and some biochemical events of them were comparatively investigated. Results： The gene slp had been deleted successfully. The mutant grew faster than wild strain at the prime period, however, the wild strain declined faster. The biochemical feature of the wild strain and the mutant is similar. Conclusion： The putative S-layer protein SLP deletion mutant of Bacillus anthracis was obtained.