定向敲除牙龈卟啉单胞菌菌毛蛋白FimA的重组质粒构建

Construction of recombinant plasmid with Porphyromonas gingivalis FimA deficiency

目的构建定向敲除牙龈卟啉单胞菌（Porphyromonasgingivalis，Pg）菌毛蛋白FimA基因的质粒pPHU281_A_Spec_B，用于后续构建菌毛蛋白FimA缺陷型Pg，为研究FimA的功能奠定分子基础。方法厌氧培养PgATCC33277，提取基因组DNA，聚合酶链反应扩增PgFimA基因一定长度的上游A片段及下游B片段，并在扩增片段两端添加合适的酶切位点；利用定向克隆技术将A和B基因插入到载体自杀质粒pPHU281中，并添加大观霉素抗性基因片段，重组的pPHU28_1_A_Spec_B在大肠杆菌DH-5仪内扩增后酶切电泳鉴定并进行DNA测序。结果通过对重组质粒pPHU281_A_Spec_B进行酶切鉴定以及DNA序列测定分析，证明重组质粒pPHU28_1_A_Spec_B构建成功。结论成功构建了质粒pPHU28_1_A_Spec_B，有利于菌毛蛋白FimA缺陷型Pg的构建，为深入研究FimA的作用机制奠定了基础。

Objective To construct the recombinant plasmid pPHU281 A_Spec_B, which knock out Porphyrmonas gingivalis（Pg） FimA gene. Methods Genomie DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments： upstream, downstream of FimA gene and speetinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5c~. Results The gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed. Conclusions The recombinant plasmid pPHU281 A Spec_B was constructed,which may be used for the constructon of FimA deficient Pg.