植物乳杆菌Lactobacillu plantarumY1菌株胆盐水解酶基因(bsh)的克隆及重组表达

Molecular Cloning and Expression of the Bile Salt Hydrolase Gene(bsh) From Lactobacillus plantarum Y1

经同源蛋白比对分析设计引物,PCR扩增获得植物乳杆菌Y1菌株bsh基因(975 bp),首先克隆至表达载体pET-28a,转化E.coli BL21(DE3)菌株,IPTG诱导表达,SDS-PAGE分析结果显示表达的重组蛋白为包涵体.选择能为大肠杆菌稀有密码子提供额外tRNA的E.coli Rosetta(DE3)作为宿主菌,仍旧没有改善表达产物的可溶性.但是,选择含IF2融合蛋白标签的pLS-IF2质粒构建表达载体,SDS-PAGE分析及Western blot鉴定结果显示表达的融合重组蛋白IF2-BSH具有可溶性.该结果为进一步研究乳酸菌胆盐水解酶的生物活性,结构功能关系的研究奠定了基础.

Based on the homologous protein BLAST result,the bsh gene of Lactobacillus plantarum Y1（975bp） was amplified by PCR and subcloned into expression vector pET-28a.The constructed recombinant plasmid pET-28a-BSH was transformed to Escherichia coli BL21（DE3）for expression under induction of IPTG.SDS-PAGE profile indicated that the expressed protein mainly existed in a form of inclusion body.The solubility of the expressed protein was not improved when choosing E.coli Rosetta（DE3） as the expression host that could supply additional tRNA for rare codons.However,when pLS-IF2 was chosen to construct the expression plasmid that contained the fusion protein tag ＂IF2＂,SDS-PAGE profile and Western blot results showed that the expressed product IF2-BSH junction to improve the solubility efficiency,respectively.Therefore,on basis of the fusion tags technology,the bsh gene of L.plantarum Y1 was successfully cloned and expressed in E.coli.,which laid a foundation of study on bioactivity and relation between structure and functions of Bile Salt Hydrolase from Lactobacillus sp.