鱼源嗜水气单胞菌外膜蛋白基因ompTS在枯草芽胞杆菌中的表达及检测  

Expression and Detection of Outer Membrane Protein Gene OmpTS of Fish Aeromonas hydrophila in Bacillus subtilis

作　　者：罗晓松[1,2] 侯化鹏[1] 尚书[1] 汪登强[2] 邹世平[1,2] 龙良启[1] 

机构地区：[1]华中农业大学动物医学院,武汉430070 [2]中国水产科学研究院长江水产研究所,荆州434000

出　　处：《农业生物技术学报》2009年第4期554-560,共7页Journal of Agricultural Biotechnology

基　　金：农业部结构调整重大技术研究专项(No.06-05-03B)资助

摘　　要：用PCR方法从枯草芽胞杆菌(Bacillus subtilis)、鱼源嗜水气单胞菌(Aeromonas hydrophila)的基因组DNA和质粒pGFP-2中,分别扩增P43启动子、不含信号肽的外膜蛋白基因ompTS和不含启动子的绿色荧光蛋白基因。各PCR产物经测序、酶切、连接到大肠杆菌(Escherichia coli)-枯草芽胞杆菌穿梭载体pNW33N相应位点分别构建了穿梭表达载体pNWP43gfp和pN-WP43omp。电转枯草芽胞杆菌构建菌株BS01(pNWP43gfp),在荧光显微镜下发出明亮的绿色荧光;电转枯草芽胞杆菌构建菌株BS01(pNWP43omp),表达的外膜蛋白经SDS-PAGE和Westernblot检测,结果表明,穿梭表达载体pNWP43gfp和pNWP43omp构建成功。构建的枯草芽胞杆菌在P43启动子的调控下,分别实现了绿色荧光蛋白基因和外膜蛋白基因ompTS的表达。Bacillus subtilis is an attractive host in bioindustry. In order to develop the application of B.subtilis in aquaculture, the shuttle expression vector pNWP43gfp and pNWP430mp were constructed,respectively. With the technology of PCR, the promoter P43 was amplified from B. subtilis total DNA, the green fluorescent protein gene without promoter sequence was amplified from pGFP-2, and the outer membrane protein gene ompTS without signal peptide-encoding sequence was amplified from A. hyclrophila total DNA. The PCR productions were sequenced, digested and cloned into the corresponding site of an E. coli-B, subtilis shuttle vector pNW33N to generate the shuttle expression vector pNWP43gfp and pNWP430mp. The recombinant vector were transformed into B. subtilis BS01, respectively. BS01 cells harboring pNWP43gfp could produce bright green fluorescence which examined by fluorescent microscopy. BS0 1 cells harboring pNWP430mp expressed the outer membrane protein, which were confirmed by SDS-PAGE and Western blot. The shuttle expression vector pNWP43gfp and pNWP430mp were successful constructed. Under the control of the promoter P43, the gfp and ompTS genes were respectively expressed in B. subtilis.

关 键 词：P43启动子 pNW33N穿梭载体 绿色荧光蛋白 外膜蛋白 嗜水气单胞菌 枯草芽胞杆菌 

分 类 号：S188[农业科学—农业基础科学]