牙龈卟啉单胞菌FtsZ降解试验模型-clpP基因缺失株的构建

目的 建立牙龈卟啉单胞菌分裂关键蛋白FtsZ （FtsZPg）体内降解试验模型,即构建蛋白酶编码基因clpP缺失的E.coli MG1655.方法 将一段两端含靶基因同源臂的PCR产物电转入L-阿拉伯糖诱导后表达λ噬菌体重组蛋白、具有较强重组能力、含质粒pKD46的菌株E.coli MG1655感受态细胞,以PCR产物中的氯霉素抗性基因替代靶基因.结果 利用氯霉素抗性平板筛选阳性重组体,得到E.coli MG1655的ClpP蛋白酶基因敲除突变株.通过琼脂糖凝胶电泳和DNA测序最终鉴定阳性clpP基因缺失株.结论 本试验成功构建了E.coli MG1655 clpP敲除株;该敲除株有望在探讨重要牙周致病菌牙龈卟啉单胞菌分裂关键蛋白FtsZPg与蛋白酶clpP的关系中发挥一定作用.

Construction of a model-clpP gene deleted strain for degradation experiments of the essential cell-division protein FtsZ in Porphyromonas gingivalis

Objective To delete clpP gene in chromosome of Escherichia coli （E. coli） MG1655 for in vivo degra-dation experiments of the essential cell - division protein FtsZ in Porphyromonas gingivalis （ FtsZPs ）. Methods PCR products were introduced into E. coli MG1655 with plasmid pKD46 by electroporation. Then the strains could express re-combination proteins from bacteriophage λ and acquire the function of recombination when induced by L - arabinose. The clpP gene was replaced by chloramphenicol resistance gene in the polymerase chain reaction （PCR） products. Results The clpP gene deleted strains expressing chloramphenicol resistance gene were selected by chloramphenicol agar. The clpP gene deleted strains were furtherly identified by PCR and DNA sequencing. Conclusion The clpP gene deleted E. coli MG1655 was constructed, which will be useful for the investigation into the relationship between the cell-division protein FtsZPs and the protease ClpP.