枯草芽孢杆菌guaB基因克隆及其在大肠杆菌中的表达

Cloning and Expression of Bacillus Subtilis guaB Gene in Escherichia Coli

克隆枯草芽孢杆菌guaB基因,构建穿梭表达载体,使其在大肠杆菌中获得表达,为构建鸟苷高产工程菌奠定基础。从枯草芽孢杆菌JSIM-G-518基因组扩增出guaB基因,将其连接到pMD19-T上,得到重组质粒pMD-guaB,进行测序和Blast比对分析。该重组质粒经PstI、BamHI双酶切,获得guaB片段连接至pHP13载体上,构建穿梭表达载体pHP13-guaB,并采用双酶切电泳进行鉴定。将该载体转化入大肠杆菌BL21中,通过SDS-PAGE电泳检测guaB基因的表达效果。克隆的guaB基因长度为1 510 bp,与GenBank登录的枯草芽孢杆菌同源性为99%,编码500个氨基酸,确认为控制IMP脱氢酶的基因。穿梭载体pHP13-guaB经双酶切后产生1 510 bp和4.9 kb两个期望的目的条带。SDS-PAGE结果显示表达产物相对分子质量的大小为52.9 k,与预期的IMP脱氢酶分子量一致,且该目的蛋白的表达效果比对照组更明显。论文成功实现了枯草芽孢杆菌guaB基因在大肠杆菌中的表达。

The guaB gene was cloned from Bacillus subtilis to construct shuttle expression vector and expressed in Escherichia coli, laying the foundation for building high-yielding guanosine engineered bacteria. Firstly, the guaB gene was amplified from the genomic DNA of Bacillus subtilis JSIM-G-518, cloned into pMD19-T vector to form the recombined plasmid pMD-guaB, and identified by sequencing and blast analysis. Secondly,the guaB gene was obtained from pMD-guaB by PstI and BamHI digestion, subcloned into the plasmid pHP13 to form the shuttle express vector pHP13-guaB, and identified by agarose gel electrophoresis with double enzyme digestion. Finally, the pHP13-guaB vector was transformed into E coli BL21, and its expression effect was analyzed by SDS-PAGE. The cloned guaB gene had 1 510 bp length, with 99% homology with Bacillus subtilis of Genbank, encoded 500 amino acids, and demonstrated to be the gene encoding the IMP dehydrogenase. After the pHpl 3-guaB was digested by pstI and BamH I, the agarose gel electrophoresis results showed that two desired target fragments were of 1 510 bp and 4. 9kb. The SDS-PAGE results showed that the expressed protein was 52. 9k molecular weight, corresponding with the expected IMP dehydrogenase, and its expressed effect was more obviously than the control sample. The results demonstrated that the Bacillus subtilis guaB gene had been expressed in Escherichia coli successfully.