嗜碱芽孢杆菌Bacillus sp. N16-5表达载体的构建  

Construction of expression vectors for alkaliphilic Bacillus sp. N16-5

作　　者：刘文侠[1,2] 李金山[1] 高成华[1] 薛燕芬[1] 马延和[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院大学,北京100049

出　　处：《微生物学报》2014年第1期89-96,共8页Acta Microbiologica Sinica

基　　金：国家"973项目"(2013CB733900);国家"863计划"(2011AA02A206)~~

摘　　要：【目的】构建可以在嗜碱芽孢杆菌Bacillus sp.N16-5中表达外源基因的表达载体。【方法】以枯草芽孢杆菌表达质粒pHCMC04为基本骨架,删除木糖诱导的启动子和木糖阻遏蛋白基因,分别插入枯草芽孢杆菌组成型强启动子P43和嗜碱芽孢杆菌N16-5的组成型启动子P EF,构建表达载体pABN165P43和pABN165P EF;将绿色荧光蛋白基因gfp作为报告基因连接至表达载体得到pABN165P43-gfp和pABN165P EF-gfp,利用原生质体转化法将其转化至Bacillus sp.N16-5,通过荧光显微镜和多功能荧光读板仪检测报告基因的表达情况。【结果】所构建的表达载体pABN165P43-gfp和pABN165P EF-gfp可以在Bacillus sp.N16-5中表达报告基因gfp,荧光定量数据显示,在7.5 h左右开始检测到绿色荧光蛋白的表达,从7.5 h到12 h荧光强度随时间增长迅速并在12 h左右达到最大,最大荧光值在7000左右。【结论】成功构建了2个嗜碱芽孢杆菌Bacillus sp.N16-5表达载体,实现了外源基因在嗜碱芽孢杆菌中的表达。[Objective]To construct an expression vector for alkaliphilic Bacillus sp.N16-5.[Methods]Bacillus subtilis expression vector pHCMC04 was used as a backbone.Its xylose-inducible promoter cassette was replaced by the constitutive promoters P43 （from B.subtilis） and PEF（from Bacillus sp. N165）,separately,resulting in two expression vectors pABN165P43 and pABN165PEF.Green fluorescent protein gene gfp was linked to the two vectors as a reporter gene.Fluorescence microscope and multifunctional fluorescent reader were used to test the expression efficiency of the system.[Results]Green fluorescence was visualized in Bacillus sp.N16-5 with pABN165PEF-gfp or pABN165P43-gfp.Quantitative data analysis revealed that fluorescence was first detected around the 7th hour.The fluorescence intensity increased rapidly from the 7th hour to the 12th hour and reached the maximum at about the 12th hour.[Conclusion]Two expression vectors for Bacillus sp.N16-5 have been constructed,allowing expression of exogenous protein in alkaliphilic Bacillus sp.N165.

关 键 词：BACILLUS SP N16-5 表达载体 嗜碱芽孢杆菌 

分 类 号：Q933[生物学—微生物学]