【摘要】 目的构建粪肠球菌心内膜炎抗原efaA基因转化株,探讨efaA基因在肠球菌生物膜形成及感染性心内膜炎致病中的作用。方法 PCR扩增粪肠球菌efaA基因,构建pAT28-efaA重组子,经PCR、双酶切及测序鉴定,将重组子转化到efaA-野生株S14做为转化株(S14-pAT28-efaA);同时将空质粒pAT28转化到野生株作为对照株(S14-pAT28);IPTG诱导efaA表达,SDS-PAGE、Western Blot分析鉴定。微量滴定板法测定生物膜形成能力。将S14、S14-pAT28、S14-pAT28-efaA分别接种到96孔板,37℃培养24h形成生物膜,结晶紫染色,测定孔板底部在595nm的OD值,比较efaA基因转化前后的生物膜形成能力。结果成功构建粪肠球菌心内膜炎抗原efaA基因转化株;转化株的OD595值大于野生株及空质粒转化株,P<0.05,空质粒转化株的OD595值与野生株比较差异无统计学意义,P>0.05。结论粪肠球菌心内膜炎抗原efaA基因有利于肠球菌生物膜的形成。更多还原

【Abstract】 In this study,we construct E.faecalis efaAtransformant in order to study the role of efaA on biofilm formation and the pathogenetic mechanism of infectious endocarditis.The efaA gene was amplified fromEnterococcus faecalis by PCR.The recombinant plasmid pAT28-efaA was confirmed by PCR,double enzyme digestion and sequencing,and then transformed into efaA-wild strain S14 as the transformant S14-pAT28-efaA;while the pAT28was transformed into wild strain S14 as a control strain S14-pAT28.Expression of efaA was induced by IPTG,and confirmed by SDS-PAGE and Western blotting. Biofilms were detected by microtiter plate assay.S14,S14-pAT28and S14-pAT28-efaA were cultivated in 96-well for 24hours; biofilms were detected by microtiter plate reader after Gram crystal violet staining.Optical density at 595nm for each strain was determined and compared.It was shown that the transformant S14-pAT28-efaA was successfully constructed.OD595 values of the transformant were greater than that of wild strain and control strain respectively(P<0.05).There was no significant difference of OD595 between control strain S14-pAT28and wild strain S14(P>0.05).It was concluded that E.faecalis efaA might be in favor of the biofilm formation.更多还原