肠球菌efaA基因阳性转化株的构建及其对生物膜形成影响的初步探讨

发布时间：2018-07-14 20:38

【摘要】： 目的:构建pAT28+efaA+His重组子和肠球菌efaA-对照株及efaA+转化株,为进一步探讨efaA基因的功能,防治肠球菌感染奠定基础。方法: 一、肠球菌efaA-对照株及efaA+转化株的构建及鉴定: 1.用PCR从肠球菌标本中筛选出efaA-/gel-/cyl-野生株(本实验室保存); 2.用氯化钙共沉淀法将空质粒pAT28转化到野生株作为对照株; 3.构建pAT28+efaA重组子并转化到野生株做为转化株:Genebank中获得efaA基因全长,PCR扩增efaA基因片段,用基因克隆技术构建pAT28+efaA重组载体,用氯化钙共沉淀法将重组子转化至E.coli DH5α,经蓝白斑筛、PCR鉴定、双酶切鉴定、测序,将测序正确的重组子转化到野生株做为转化株, IPTG诱导efaA基因表达, SDS-PAGE、Western Blot分析鉴定。二、efaA基因对肠球菌生物膜形成影响的初步探讨用半定量可见光测定OD值的方法检测efaA-野生株、空质粒pAT28对照株以及efaA+转化株(大约5×106-10×106 CFU)在96孔板分别静止培养3 h、24 h后形成的生物膜OD595 ,通过比较efaA基因转化入肠球菌野生株前后生物膜形成能力差异,探讨efaA基因对生物膜形成的影响。结果:1.对照株(空质粒pAT28转野生株)在含壮观霉素(终浓度500μg/ml)的ELB培养平板上培养可见菌落生长;转化株(pAT28+efaA重组子转野生株)在IPTG/X-Gal的壮观霉素(终浓度500μg/ml) ELB培养平板上培养,可见白色菌落生长,经IPTG诱导表达,SDS-PAGE、Western Blot分析,可见在35 kd附近有一条带;2.早期黏附测定与生物膜形成实验中,转化株粘附在96-孔板底部形成的生物膜OD595值均比野生株及对照株大。结论:1.成功构建出肠球菌efaA-对照株及efaA+转化株;2. efaA基因有利于肠球菌早期黏附与生物膜形成。
[Abstract]:Objective: to construct pAT28 efaA his recombinant strain, Escherichia coli EFA A- control strain and efaA transformation strain, so as to further study the function of efaA gene and lay a foundation for prevention and treatment of Enterococcus infection. Methods: 1. Construction and identification of Escherichia coli EFA A-control strain and efaA transformation strain: 1. The wild strains of EFA-r-gel-r-cyl- (preserved in our laboratory) were screened by PCR from Enterococcus specimens; 2. The empty plasmid pAT28 was transformed into wild strain by calcium chloride coprecipitation method. The recombinant plasmid pAT28 efaA was constructed and transformed into wild strain as strain: Genebank. The full-length efaA gene fragment was amplified by PCR. The recombinant vector pAT28 efaA was constructed by gene cloning technique. The recombinant plasmid was transformed into E. coli DH5 伪 by calcium chloride coprecipitation. The recombinant was identified by PCR, double enzyme digestion and sequencing. The recombinant was transformed into wild strain as transformed strain. IPTG induced efaA gene expression and identified by SDS-PAGEG Western blot. Preliminary study on the effect of DianefaA gene on the biofilm formation of Enterococcus spp. The OD value of Escherichia coli was measured by semi-quantitative visible light. Blank plasmid pAT28 control strain and efaA transformation strain (about 5 脳 106-10 脳 106CFU) were cultured at 96-well plate for 3 h or 24 h respectively to form biofilm OD595. The ability of biofilm formation was compared before and after efaA gene was transformed into wild strains of Enterococcus. To investigate the effect of efaA gene on biofilm formation. The result is 1: 1. The control strain (blank plasmid pAT28 transformed wild strain) was cultured on the ELB plate containing spectinomycin (final concentration 500 渭 g/ml), and the transformed strain (pAT28 efaA recombinant transformed wild strain) was cultured on the IPTG- X-Gal spectinomycin (final concentration 500 渭 g/ml) ELB plate. The growth of white colony and the expression of SDS-PAGEG by IPTG were analyzed by Western blot, and a band 2 was found near 35kd. In the early adhesion assay and biofilm formation test, the OD595 values of the biofilm formed at the bottom of the 96-hole plate were larger than those of the wild strain and the control strain. Conclusion 1. E.coli EFA-control strain and efaA transformation strain were successfully constructed. The efaA gene was beneficial to the early adhesion and biofilm formation of Enterococcus.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.1

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