摘要
目的:构建重组质粒pPHU281-C-Spec-E,用于敲除菌毛蛋白次要组分基因fimCDE。构建fimCDE缺陷的牙龈卟啉单胞菌,为进一步研究FimCDE在感染中的作用奠定基础。方法:厌氧罐培养牙龈卟啉单胞菌菌株ATCC33277,提取基因组DNA后,PCR扩增获得含有人工设计的酶切位点的fimC基因的上游片段及fimE的下游片段,将其克隆到自杀质粒pPHU281内,命名为pPHU281-C-E;再将抗大观霉素的抗性基因插入到C及E片段之间,最终获得重组质粒pPHU281-C-Spec-E。结果:酶切及DNA序列分析验证重组质粒pPHU281-C-Spec-E构建成功。结论:成功构建了含有牙龈卟啉单胞菌菌毛蛋白次要组分基因fimC上游及fimE下游片段的重组质粒pPHU281-CSpec-E,可用于构建菌毛蛋白次要组分FimCDE缺陷的的突变体。
PURPOSE： To construct a recombinant plasmid containing the upstream offimC and downstream of timE of Porphyromonos gingivalis, designated as pPHU281-C-Spec-E, which may be further used to knock outfimCDE gene to determine the role of FimCDE in the infection by P. gingivalis. METHODS： DNA fragments were generated by PCR with the genomic DNA of P. gingivalis strain ATCC 33277 as the template. The upstream fragment offimC （fragment C） and downstream fragment of timE （fragment E） were cloned into the suicide plasmid pPHU281 to generate plasmid pPHU281- C-E. The spectinomycin resistance gene was inserted between fragment C and E to construct plasmid Pphu281-C-Spec- E. The recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. RESULTS： The recombinant plasmid pPHU281-C-Spec-E was successfully constructed, which was ready for generation of FimCDE- knockout mutant of P. gingivalis. CONCLUSIONS： The recombinant plasmid pPHU281-C-Spec-E is a tool for construction of FimCDE deficient mutant of t9. gingivalis. Supported by National Natural Science Foundation of China （81070839）. Team Project of Medical Leaders in Talent and Innovation of Jiangsu Province （I3201110）, Science and Technology Development Plan of Nanjing City （YKK06115） and Medical Science and Technology Development Project of Nanjing City（ZKX1030）.
出处
《上海口腔医学》
CAS
CSCD
北大核心
2013年第6期623-627,共5页
Shanghai Journal of Stomatology
基金
国家自然科学基金(81070839)
江苏省医学领军人才与创新团队项目(LJ201110)
南京市科技发展计划项目(YKK06115)
南京市医学科技发展重点项目(ZKX1030)~~
