摘要
【背景】沙门氏菌是一种重要的人畜共患病原菌,可引起广泛的胃肠炎以及伤寒、副伤寒,其致病机制一直未被阐明。基因敲除技术在研究沙门氏菌致病性方面发挥了重要作用,然而目前的敲除技术仍存在费时、成功率低的问题。研究发现鼠伤寒沙门氏菌含有Ⅵ型分泌系统,其组成成分之一溶血素共调节蛋白(Hemolysin-coregulated protein,Hcp)可能在其致病过程中发挥了重要作用。【目的】拟通过对3个编码Hcp蛋白的基因进行敲除,在鼠伤寒沙门氏菌中建立一套方便快捷的重组系统,从而用于沙门氏菌致病性的研究。【方法】以pKD4为模板,扩增两端带有目的基因同源序列的卡那霉素抗性基因片段,将片段导入含重组酶系统的目的菌,重组后再导入质粒pCP20消除抗性基因片段,达到无痕敲除的效果。【结果】对3个单独的hcp基因及其组合进行了敲除,得到了所需的基因缺失株,并总结出了一些实验过程中可能遇到的问题的解决方案。【结论】Red重组系统可用于鼠伤寒沙门菌的基因敲除,通过优化同源片段的长度、PCR模板浓度、L-阿拉伯糖加入时间、实验过程中的温度等实验条件,提高Red重组系统在沙门氏菌中的重组效率。此方法简单、快速,重组效率高,值得推广。
[Background] Salmonella is an important food-borne pathogen causing disease such as gastroenteritis, typhoid fever and paratyphoid fever in humans and animals worldwide. The pathogenic mechanism is still unclear. Gene knockout plays an important role in studying the pathogenicity of Salmonella, however, currently it is time consuming and the success rate is very low. S. typhimurium has a Type Ⅵ secretion system（T6 SS）. The component Hemolysin-coregulated protein（Hcp） may play an important role in its pathogenicity. [Objective] To establish a quick and effective gene knock out system by knocking out the three Hcp encoding genes in S. typhimurium, thus to study the pathogenicity of Salmonella. [Methods] Kanamycin resistance gene fragments with homologous upstream and downstream sequences of hcp genes were amplified with pKD4 as template, and then were introduced into S. typhimurium which has Red recombination system enzyme. Then pCP20 were electroporated into the cells to delete the integrated kanamycin resistant gene from the recombinant bacteria. [Results] Both individual hcp genes and their recombinations were successfully knocked out from the genome of S. typhimurium. We also summarized some solutions to the problems we may encounter. [Conclusion] Red recombination system is a good method to knock out genes in S. typhimurium. We can improve the efficiency by optimizing the experimental conditions such as the length of the homologous fragment, the concentration of the PCR templates, the time point for L-arabinose addition and the culture temperature. It is a simple and efficient method and deserves to be popularized.
作者
王萍
董俊芳
邹清华
WANG Ping;DONG Jun-Fang;ZOU Qing-Hua(Department of Microbiology ＆ Infectious Disease Center,School of Basic Medical Sciences,Peking University Health Science Center,Beijing 100191,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2018年第11期2529-2536,共8页
Microbiology China
基金
国家自然科学基金(81572041)~~
关键词
鼠伤寒沙门菌
Ⅵ型分泌系统
溶血素共调节蛋白
RED重组系统
Salmonella typhimurium
Type Ⅵ secretion system （T6SS）
Hemolysin-coregulated protein （Hcp）
Red recombination system
