甲型副伤寒沙门氏菌ompW基因功能的初步研究  

Preliminary study of functions of ompW gene in Salmonella paratyphi A

作　　者：陈沁[1,2] 李娜[1] 梁吴宇[1] 王斌[1] 魏华[2] 曾明[1] 

机构地区：[1]中国食品药品检定研究院,卫生部生物技术产品检定方法及其标准化重点实验室,北京100050 [2]南昌大学食品科学与技术国家重点实验室,330047

出　　处：《中华微生物学和免疫学杂志》2012年第1期6-11,共6页Chinese Journal of Microbiology and Immunology

基　　金：863计划资助课题（2006AA02A236）;国家自然科学基金资助项目（81071325）

摘　　要：目的利用λRed重组系统敲除甲型副伤寒沙门氏菌ompW基因构建突变株，并且制备相应的回复株，进而初步研究该基因的功能。方法PCR扩增得到上、下游同源臂，与卡那霉素抗性基因片段共同构建打靶片段，将其浓缩后转化含bRed重组系统的甲型副伤寒沙门氏菌（50973株），经PCR鉴定后得到突变株；将重组酶表达质粒pACUl84与ompW基因的调控区和编码区序列连接后电转入突变株中，双酶切鉴定得到相应的回复株；SDS．PAGE及Western blot鉴定野生株、突变株与回复株中OmpW蛋白的表达情况；生化鉴定野生株、突变株与回复株，并观察野生株与突变株生长曲线的差异；测定野生株、突变株与回复株活菌半数致死量（LD50），以此来观察ompW基因与细菌毒力的相关性。结果在甲型副伤寒沙门氏菌50973株中成功敲除了ompW基因，并构建了相应回复株；野生株与回复株均可表达出OmpW蛋白，突变株没有出现该蛋白的表达；野生株、突变株与回复株生化鉴定结果均为甲型副伤寒沙门氏菌，且野生株和突变株生长无明显差异；三者LD50均不存在显著差异。结论甲型副伤寒沙门氏菌的ompW基因与该菌致病毒力无相关性，但突变株的构建为后续研究该基因具体功能提供了基础。Objective To construct ompW- and ompW＋ mutants of Salmonella paratyphi A with kRed system, and then study the function of the gene preliminarily. Methods Homologous regions were amplified from the genome Salmonella paratyphi A 50973, and then connect with kana fragment from plasmid pET22b-kan to construct a recombinant vector. The resultant fragments were amplified and transferred into 50973 with the help of XRed system after its concentration. Then the ompW- mutant was obtained PCR iden-tification. Connect the recombinase expression plasmid pACU184 with full fragment of ompW regulatory region and coding region, then transfer the connection product into the mutant, the ompW＋ mutant was obtained after double digest identification. Full cells of the wild, ompW- and ompW＋ mutants were samples for SDS-PAGE and Western blot to detect the expression of protein OmpW. Biochemical identification of wild strain and mutant strains was conducted, so did the growth curves of the wild and the ompW- mutant. Choose BALB/c mice as a model to determine median lethal dose LD50 of the wild and mutant strains in order to observe the correlation between ompW gene and bacterial virulence. Results ompW gene was knocked out in Salmonella paratyphi A 50973, also the ompW＋ mutant was constructed ; The wild and ompW＋ mutant express the protein OmpW, while the ompW- mutant lost the protein. Each of the wild and mutant strains was Salmonella paratyphi A, and no obvious difference could be observed for their growth curves. LDs0 for each strain was also similar. Conclusion The ompW gene has no correlation with the virulence in S. paratyphi A 50973, but the contribution of the mutants made an important foundation for the further study of functions of the gene ompW in Salmonella paratyphi.

关 键 词：甲型副伤寒沙门氏菌 ompW 基因敲除 功能研究 

分 类 号：R378[医药卫生—病原生物学]