【摘要】 目的原核表达日本血吸虫乳酸脱氢酶(SjLDH),对表达产物进行纯化及生物活性鉴定。方法将SjLDH编码序列克隆入pET-28a原核表达载体并转染大肠埃希菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后亲和层析法对表达产物进行纯化,并用聚丙烯酰胺变性凝胶电泳(SDS-PAGE)、蛋白印迹(Western blot)、酶联免疫吸附实验(ELISA)、酶活性染色及活性测定进行鉴定。结果成功构建pET28a-SjLDH重组质粒并在大肠埃希菌中获得可溶性表达,亲和层析纯化表达产物。SDS-PAGE及Western Blot结果显示,表达及纯化产物分子量约36kDa,与SjLDH理论分子量相符。ELISA结果表明,重组蛋白具有良好的免疫活性,酶活性染色表明重组蛋白具有乳酸脱氢酶活性,活性单位为379U/mg。结论成功进行SjLDH的原核表达并获得纯化的重组蛋白,重组蛋白具有良好的免疫原性及酶活性,为研究SjLDH的功能奠定了基础。更多还原

【Abstract】 Objective To express SjLDH in prokaryotic expression system,purify and identify the protein expressed.Methods The encoding fragment of SjLDH was inserted into plasmid pET-28a and the recombiant plasmid pET-28a-SjLDH was transformed into E.coli BL21 and induced by IPTG.The expressed products were purified by affinity chromatography column and identified by SDS-PAGE,Western blot,ELISA,enzymatic vital staining and enzymatic activity detection.Results Recombinant plasmid pET-28a-SjLDH was constructed and expressed in E.coli successfully.A 36-kDa protein with 6×His-tag was purified by affinity column and identified by SDS-PAGE and Western blot,which had high LDH activity of 379 U/mg and high immunological activity.Conclusion Recombinant SjLDH was obtained successfully,which had high enzymatic activity and immunological activity.This work provided the essential material for the functional study on SjLDH.更多还原