生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2012年
6期
777-780
,共4页
冉金宝%廖翔%周围%王羽%魏波%高原%岳俊杰%梁龙%王纯洁
冉金寶%廖翔%週圍%王羽%魏波%高原%嶽俊傑%樑龍%王純潔
염금보%료상%주위%왕우%위파%고원%악준걸%량룡%왕순길
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
膜蛋白複閤物%缺失突變株%重疊延伸 PCR%λRed 重組繫統%弗氏誌賀菌 M90T 株
막단백복합물%결실돌변주%중첩연신 PCR%λRed 중조계통%불씨지하균 M90T 주
membrane protein complex%gene knock-out mutant%overlap extension PCR%λRed recombination system%Shigella flexneri M90T
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.
目的:構建弗氏誌賀菌 M90T 株毒力相關膜蛋白複閤物組成蛋白基因的缺失突變株.方法:分彆擴增目的基因的上、下遊同源臂和卡那黴素抗性基因片段,利用重疊延伸 PCR 技術將這3箇片段融閤為打靶片段,電擊轉化M90T/pKD46感受態細胞,在λRed 重組繫統的作用下,通過同源重組將目的基因置換為卡那黴素抗性基因,之後在輔助質粒 pCP20的作用下切除 FRT 位點之間的卡那黴素抗性基因,得到基因缺失突變體.結果與結論:分彆構建瞭M90T 株毒力相關膜蛋白複閤物4箇組成蛋白 Apy、DnaK、ClpB、YdgA 的基因缺失突變體,為進一步研究各基因的功能提供瞭突變體.
목적:구건불씨지하균 M90T 주독력상관막단백복합물조성단백기인적결실돌변주.방법:분별확증목적기인적상、하유동원비화잡나매소항성기인편단,이용중첩연신 PCR 기술장저3개편단융합위타파편단,전격전화M90T/pKD46감수태세포,재λRed 중조계통적작용하,통과동원중조장목적기인치환위잡나매소항성기인,지후재보조질립 pCP20적작용하절제 FRT 위점지간적잡나매소항성기인,득도기인결실돌변체.결과여결론:분별구건료M90T 주독력상관막단백복합물4개조성단백 Apy、DnaK、ClpB、YdgA 적기인결실돌변체,위진일보연구각기인적공능제공료돌변체.
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 λRed 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.
