大肠埃希菌MG1655 Lon蛋白酶编码基因敲除株的构建  

Construction of a lon knockout strain of Escherichia coli MG1655

作　　者：韦艳霞[1] 李阳[1] 刘佃滨[2] 

机构地区：[1]徐州医学院病原生物学与免疫学教研室,江苏徐州221004 [2]徐州医学院口腔医学院

出　　处：《徐州医学院学报》2014年第7期471-473,共3页Acta Academiae Medicinae Xuzhou

基　　金：国家自然科学基金（31300029）;江苏省自然科学基金（BK20130213）;江苏省高校“青蓝工程”优秀青年骨干教师项目;徐州医学院优秀人才科研启动基金（D2012014）

摘　　要：目的构建Lon蛋白酶编码基因缺失的大肠埃希菌（E．coli MG1655）菌株。方法PCR扩增基因序列，该序列两端与Lon蛋白酶基因部分序列同源，中间部分为氯霉素抗性基因。电转化法将该片段转入大肠埃希菌MG1655。利用宿主菌-大肠埃希菌MG1655内pKIM6编码的入重组系统，以PCR产物中的氯霉素抗性基因替代靶基因-Lon编码基因。氯霉素抗性平板筛选阳性重组体，琼脂糖凝胶电泳和DNA测序两种方法鉴定lon敲除突变株。结果氯霉素抗性平板成功筛选出重组菌株，基因检测结果表明Lon编码基因被氯霉素抗性基因替代。结论本研究通过该方法成功获得了E．coliMG1655蛋白酶编码基因lon敲除突变株，为探讨大肠埃希菌Lon在牙菌斑形成中的作用奠定了基础。Objective To establish an E. coli MG1655 strain lacking lon gene. Methods A gene sequence was amplified by PCR, which contained the flank sequences of lon and the chloramphenicol resistance gene located between the flank sequences. Then, the resultant fragments were introduced into E. coli MG1655 by electroporation. The lon was replaced by the chloramphenicol resistance gene, when a bacteriophage X recombination system on plasmid pKD46 in E. coli MG1655 was induced by L - arabinose. The lon knockout strain was identified by polymerase chain reaction （PCR） and DNA sequencing. Results The lon knockout strain was successfully screened out by chloramphenicol agar. The results of PCR and DNA sequencing showed that the lon was replaced by the cbloramphenicol resistance gene. Conclusion The Ion knockout strain of E. coli MG1655 has been established, which can facilitate the research about the role that the lon gene takes in the formation of dental Dlaoue.

关 键 词：Lon蛋白酶 基因敲除 大肠埃希菌 牙菌斑 

分 类 号：R781.4[医药卫生—口腔医学]