小鼠胆盐依赖脂肪酶蛋白原核表达载体的构建与蛋白制备

摘要

目的 构建小鼠胆盐依赖脂肪酶(BSDL)重组蛋白的大肠埃希菌原核表达载体,制备具有生物学活性的小鼠BSDL蛋白.方法 首先应用PCR合成mBSDL的cDNA序列,克隆至原核表达载体pET-28b,转化大肠埃希菌表达菌种BL21 (DE3)感受态细胞,IPTG诱导重组蛋白表达.使用His Trap FF crude柱纯化重组mBSDL蛋白.Western blot鉴定后采用尿素浓度梯度降低法进行透析复性.结果 成功构建原核表达载体pET-28b-mBSDL,实现该蛋白在E.coli BL21 (DE3)表达菌种中的高效表达,并且表达蛋白主要存在于包涵体中.Western blot示获得较好的纯化效果且纯化后成功复性.结论 成功构建mBSDL基因的原核表达质粒并制备具有活性功能的胆盐依赖脂肪酶,为进一步研究该蛋白的功能奠定了基础.%Objective To construct a prokaryotic expression system for mBSDL in E.coli BL21 (DE3) cells and prepare the active mBSDL protein.Methods mBSDL cDNA was amplifed via PCR and inserted into the prokaryotic expression vector pET-28b.The expression of recombinant protein was induced by IPTG in E.coli BL21 cells.Then it was purified via His Trap FF crude purification system and refolded via dialysis system.The specificity of mBSDL protein was analyzed by Western blot.Results The prokaryotic expression vector pET-28b-mBSDL expressing mBSDL protein was successfully constructed and transformed into E.coli BL21(DE3) cells.The mBSDL protein was present as insoluble inclusion bodies in the bacterial extract.Western blot reveled that the purification of this recombinant protein was successful.Conclusion mBSDL protein in E.coli prokaryotic expression system is established successfully.The study provides some foundation for the further analysis of mBSDL protein.