重组胰高血糖素样肽-1载体的构建及其在枯草芽孢杆菌中的表达

Construction of expression vector of modified glucagon-like peoptide-1 and its expression in Bacillus subtilis

目的:构建及鉴定携带His标签的重组胰高血糖素样肽-1(modified glucagon-like peptide-1,mGLP-1)表达载体,实现其在枯草芽孢杆菌WB800N中的分泌表达。方法:经化学合成得到含有His标签及BamHⅠ/XbaⅠ酶切位点的mGLP-1基因片段,将其酶切后插入含信号肽amy Q序列的大肠埃希菌-枯草芽孢杆菌双穿梭载体pHT43中,得到重组载体pHT43-mGLP-1;通过电转化方法将质粒pHT43-mGLP-1转化到枯草芽孢杆菌WB800N中,构建含有目的基因的重组菌株WB800N/pHT43-mGLP-1,利用聚合酶链式反应(PCR)、碱基测序、甘氨酸聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)及蛋白质印迹法鉴定重组的枯草芽孢杆菌。结果:菌落PCR和测序结果表明,阳性克隆质粒pHT43-mGLP-1成功转化进枯草芽孢杆菌WB800N;Tricine-SDS-PAGE和蛋白质印迹法检测表明,重组菌株WB800N/mGLP-1培养上清液中有mGLP-1蛋白表达,且1 mmol/L IPTG诱导下蛋白表达量明显增加。结论:成功构建了携带mGLP-1基因的表达载体并实现了其蛋白在枯草芽孢杆菌中的分泌表达,为后续动物实验和制备微生态活菌制剂奠定了基础。

Objective:To construct and identify the expression vector expressing the protein of modified glucagon-like peptide-1(mGLP-1)containing His-tag in Bacillus subtilis WB800 N.Methods:The mGLP-1 gene including of His-tag and Bam HⅠ/XbaⅠrestriction enzyme cutting sites was obtained by chemical synthesis,and then inserted into the Escherichia coli-Bacillus subtilis shuttle vector pHT43 carrying the signal peptide amyQ sequence to construct the recombinant plasmid pHT43-mGLP-1.The plasmid pHT43-mGLP-1 was transformed into Bacillus subtilis WB800 N by electroporation.Colony PCR,sequencing technologies,Tricine SDS-PAGE and Western blotting were used to identify the recombinant Bacillus subtilis WB800 N/pHT43-GLP-1.Results:The result of colony PCR and sequencing showed that the recombinant plasmid pHT43-mGLP-1 was successfully transformed into Bacillus subtilis WB800 N.Tricine SDS-PAGE and Western blotting showed the transformed bacterium successfully expressed and released the target protein mGLP-1 to culture supernatant,and the protein expression was significantly increased by the induction of 1 mmol/L IPTG.Conclusion:The recombinant expression vector carrying mGLP-1 was successfully constructed and target protein could be expressed in Bacillus subtilis WB800 N,which will be benefical for further explore the animal experiments and micro-organism preparations.