CAJ | 학술논문

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pKD46编码的λ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．coli MG1655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。
목적：구건Lon단백매편마기인결실적대장애희균（ E．coli MG1655）균주。방법 PCR확증기인서렬，해서렬량단여Lon단백매기인부분서렬동원，중간부분위록매소항성기인。전전화법장해편단전입대장애희균MG1655。이용숙주균-대장애희균MG1655내pKD46편마적λ중조계통，이PCR산물중적록매소항성기인체대파기인-Lon편마기인。록매소항성평판사선양성중조체，경지당응효전영화DNA측서량충방법감정lon고제돌변주。결과록매소항성평판성공사선출중조균주，기인검측결과표명Lon편마기인피록매소항성기인체대。결론본연구통과해방법성공획득료E．coli MG1655단백매편마기인lon고제돌변주，위탐토대장애희균Lon재아균반형성중적작용전정료기출。
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 λrecombination system on plasmid pKD 46 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 re-sults of PCR and DNA sequencing showed that the lon was replaced by the chloramphenicol resistance gene .Conclusion The lon 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 plaque .