CAJ | 학술논문

目的构建定向敲除牙龈卟啉单胞菌(Porphyromonas gingivalis,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pPHU281 _A_Spec_B 在大肠杆菌DH-5α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pPHU281_A_Spec_B构建成功.结论成功构建了质粒pPHU281_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.
목적 구건정향고제아간계람단포균(Porphyromonas gingivalis,Pg)균모단백FimA기인적질립pPHU281_A_Spec_B,용우후속구건균모단백FimA결함형Pg,위연구FimA적공능전정분자기출.방법 염양배양PgATCC33277,제취기인조DNA,취합매련반응확증PgFimA기인일정장도적상유A편단급하유B편단,병재확증편단량단첨가합괄적매절위점；이용정향극륭기술장A화B 기인삽입도재체자살질립pPHU281중,병첨가대관매소항성기인편단,중조적pPHU281 _A_Spec_B 재대장간균DH-5α내확증후매절전영감정병진행DNA측서.결과 통과대중조질립pPHU281_A_Spec_B진행매절감정이급DNA서렬측정분석,증명중조질립pPHU281_A_Spec_B구건성공.결론 성공구건료질립pPHU281_A_Spec_B,유리우균모단백FimA결함형Pg적구건,위심입연구FimA적작용궤제전정료기출.
Objective To construct the recombinant plasmid pPHU281 A Spec_B,which knock out Porphyrmonas gingivalis(Pg) FimA gene.Methods Genomic DNA was extracted from PgATCC33277which 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 spectinomycin resistance gene.The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α.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.