日本血吸虫乳酸脱氢酶原核表达、纯化及鉴定

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。

Prokaryotic expression,purification and identification of lactate dehydrogenase from schistosome japonicum

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-28aSj LDH 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 x 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 Sj LDH was obtained successfully, which had high enzymatic activity and immunological activity. This work provided the essential material for the functional study on Sj LDH.