Rluc报告基因在大肠杆菌中的表达及检测条件优化  

Expression and Optimization of Detection of Reporter Renilla Luciferase in E.coli

作　　者：刘广文[1] 董杰[1] 闫梅英[2] 苏旭[1] 井良义[1] 郭凤华[3] 阚飙 

机构地区：[1]天津市疾病预防控制中心病原生物检测所,天津300011 [2]中国疾病预防控制中心传染病预防控制所 [3]南开大学医学院

出　　处：《环境与健康杂志》2011年第4期299-301,F0003,共4页Journal of Environment and Health

基　　金：国家自然科学基金(30700678);天津市卫生局科技基金(09KY19)

摘　　要：目的研究海肾荧光素酶报告基因(Rluc)在大肠杆菌中的表达情况及优化检测条件。方法将Rluc克隆入大肠杆菌JM109,提取质粒后用EcoRⅠ和SalⅠ进行双酶切,并与制备好的pET-30a(+)载体连接,再转化入大肠杆菌BL21。然后用ViviRen底物检测不同稀释液、不同浓度及时间等情况下重组克隆子中Rluc报告基因的荧光表达量,绘制表达曲线。结果成功构建了Rluc在BL21中的表达克隆子;ViviRen底物用PBS稀释比用LB稀释的检测灵敏度高约2倍;荧光检测的表达曲线在底物加入后迅速上升,至10 min时达高峰,然后缓慢下降;阳性克隆子中Rluc的荧光表达线性检测范围广,阴性对照菌株中背景低;总结出了ViviRen底物用PBS稀释、荧光信号在10 min后进行瞬时测定等优化检测条件。结论 Rluc报告基因可在大肠杆菌内高效表达,检测方法具有特异性好、灵敏度高等特点。Objective To study the expression and optimization of detection of reporter Renilla luciferase（Rluc） in E.coli.Methods The reporter gene Rluc was transformed into E.coli JM109 and the plasmid of clones was extracted.The plasmid was digested by restriction enzymes EcoRⅠ and SalⅠ.The fragments of Rluc purified from digested product were ligated with vector pET-30a（＋） and transformed to E.coli BL21.Fluorescence expression of reporter Rluc of the recombinant clones were detected with the ViviRen live cell substrate in different conditions,including different dilution,concentration and time.The fluorescence expression curves of Rluc were drawn.Results Recombinant clones with reporter gene Rluc in BL21 were constructed successfully.The detection level of ViviRen substrate diluted with PBS was two times as more sensitive as that diluted with LB.Expression curves showed that the fluorescence levels increased quickly and reached a peak value at the 10th minute after substrate added,and then decreased slowly.The range of detection of Rluc in recombinant clones was extensive and the background in negative control was lower.Conditions of fluorescence detection with ViviRen substrate were optimized： dilute with PBS,detect after 10 minute,etc.Conclusion Reporter gene Rluc can be successfully expressed in E.coli and its detection level is higher in sensitivity and specificity.

关 键 词：海肾荧光素酶 BL21 表达 优化 

分 类 号：R117[医药卫生—公共卫生与预防医学]