利用λRed 重组系统敲除鼠伤寒沙门氏菌sopB基因

生物技术通报 ›› 2014, Vol. 0 ›› Issue (9): 171-177.

利用λRed 重组系统敲除鼠伤寒沙门氏菌sopB基因

李晔¹,张西轩¹,郭梦征²,王素英¹,张坤生¹,阮海华¹

1. 天津商业大学生物技术与食品科学学院天津市食品生物技术重点实验室, 天津 300134;
2. 中国医学科学院放射医学研究所, 天津 300192

收稿日期:2014-04-29 出版日期:2014-09-15 发布日期:2014-09-07
作者简介:李晔，女，硕士研究生，研究方向：微生物与发酵；E-mail：liye900315@163.com
基金资助:
国家自然科学基金项目（81101220），天津市应用基础与前沿研究计划项目（12JCQNJC08100），“ 十二五”天津市中青年骨干创新人才支持计划，天津市创新团队建设项目（TD12-5049）

Deletion of sopB Gene of Salmonella typhimurium LT2 by λRed Recombination System

Li Ye¹,Zhang Xixuan¹,Guo Mengzheng², Wang Suying¹, Zhang Kunsheng¹,Ruan Haihua¹

1. Tianjin Key Laboratory of Food Science and Biotechnology，College of Biotechnology and Food Science，Tianjin University of Commerce，Tianjin 300134;
2. Institute of Radiation Medicine，Chinese Academy of Medical Sciences and Peking Union Medical College，Tianjin 300192

Received:2014-04-29 Published:2014-09-15 Online:2014-09-07

摘要/Abstract

摘要： 摘要：利用λRed重组系统敲除鼠伤寒沙门氏菌LT2的（SalmonellaentericaserovartyphimuriumLT2，S.typhimuriumLT2）sopB基因。以pKD4质粒为模板，扩增得到中间带有卡那霉素抗性基因且两端各带有59bp分别与sopB基因上下游序列同源的同源打靶片段，将其转化至表达Red重组酶的S.typhimuriumLT2感受态细胞中；在抗生素压力和λRed重组系统帮助下，同源片段和菌体sopB基因发生同源重组，通过卡那霉素筛选得到带有抗性标记的阳性重组菌；转入重组酶表达质粒pCP20以除去抗性标记，得到保留单一FRT位点的突变菌株；利用PCR技术鉴定重组菌，并通过检测沙门氏菌效应蛋白SopB的分泌以及沙门氏菌感染HeLa细胞后pAKT的激活反应来鉴定sopB基因是否被敲除。构建的ΔsopB突变菌株失去了分泌SopB蛋白的能力，且不能够像野生型菌株那样在感染HeLa细胞的过程中激活pAkt。本研究获得了S.typhimuriumLT2的sopB基因缺失突变株，为沙门氏菌感染宿主过程中SopB的功能研究提供工具，同时也为进一步探索其他类型细菌的基因敲除提供了线索。

关键词: 鼠伤寒沙门氏菌LT2, λRed, 重组系统, sopB, 基因敲除, 同源重组

Abstract: In order to investigate the role of SopB effector secreted by Salmonella typhimurium LT2, the sopB gene deletion mutant with λ Red recombination system was constructed. To amplify the homologous fragment for sopB deletion, a pair of knockout primer was designed according to the full-length sequence of sopB gene of Salmonella typhimurium LT2. With pKD4 plasmid as template, the homologous fragments including homologous regions which are similar to sopB gene upstream sequence and downstream sequence respectively and kanamycin cassette with two FRT sites were amplified. Then, the homologous fragments were electrotransformed into the Red-induced S.Typhimurium LT2 competent cells. Under the pressure of antibiotic and with the work of λ Red system, homologous recombination occurred between the fragments and genome of host strain, and the recombinants were selected on kanamycin agar plate. After the resultant recombinants were verified with PCR, the positive recombinants were cultured at 43℃overnight to eliminate pKD46. To remove the kanamycin resistant relevant DNA fragment, FLP recombinase-encoding plasmid pCP20 was introduced into the recombinants, resulting in a single FRT site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR. The removal of sopB was further verified by the secretion of SopB protein and the induction of pAkt activation in HeLa cells upon S. typhimurium LT2 infection. Considering the results including genome PCR, SopB secretion and pAkt activation in HeLa cells upon infection, it was confirmed that the sopB gene was successfully knocked out from S. typhimurium LT2 genome with λ Red recombination system. In summary, it is concluded that the sopB gene of S. typhimurium LT2 could be successfully knocked out. Moreover, this paper provides a powerful tool for the functional study of SopB effectors during the interaction between Salmonella and host. Also, it supplies the clue for the gene knock-out of other types of bacteria.

Key words: Salmonella typhimurium LT2, λ Red recombination system, sopB gene, Gene knock-out, Homologous recombination

李晔,张西轩,郭梦征,王素英,张坤生,阮海华. 利用λRed 重组系统敲除鼠伤寒沙门氏菌sopB基因[J]. 生物技术通报, 2014, 0(9): 171-177.

Li Ye,Zhang Xixuan,Guo Mengzheng, Wang Suying, Zhang Kunsheng,Ruan Haihua. Deletion of sopB Gene of Salmonella typhimurium LT2 by λRed Recombination System[J]. Biotechnology Bulletin, 2014, 0(9): 171-177.

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