人钠/二羧基转运蛋白2融合蛋白载体的构建及其原核表达

目的：观测和鉴定人钠／二羧基转运蛋白2(hSDCT2)在大肠杆菌中的表达，并分离、纯化其蛋白产物。方法：应用：DNA重组技术，构建重组表达质粒pGEX-hSDCT2；IPTG诱导其表达。采用谷胱甘肽偶联的Sepharose4B亲和层析纯化GST-hSDCT2，经Factor Xa酶切和二次亲和层析后，采用聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western Blotting进行鉴定。结果：成功构建hSDCT2基因融合蛋白载体，SDS-PAGE及Western Blotting显示GST-hSDCT2融合蛋白表达于原核细胞，蛋白产量约占菌体总蛋白量的25％。经酶切及二次纯化后，获得纯化的hSDCT2蛋白。结论：人钠／二羧基转运蛋白2可在大肠杆菌中稳定表达。

Construction of human sodium-dependent dicarboxylate transporter 2 fusion protein vector and its expression in E.coli

Objective: To observe and indentify the expression of human sodium-dependent dicarboxylate transporter 2 (hSDCT2) gene in E.coli, and isolate and purify its protein product.Methods: With DNA recombinant technique, the recombinant expressive plasmide pGEX-hSDCT2 was constructed and introduced into E.coli. GST-hSDCT2 fusion protein was expressed at the induction of IPTG. After GST-hSDCT2 fusion protein was purified by GST Sepharose 4B affinity chromatography, purified fusion protein was cleaved by Factor Xa and purified by second affinity chromatography. The results were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting assay. Results: Recombinant plasmid pGEX-hSDCT2 was successfully constructed. The results of SDS-PAGE and Western blotting assay showed (1) GST-hSDCT2 fusion protein was expressed in E.coli cell, and was produced at a level of 25% of the total cellular protein. (2) After Factor Xa cleavage and second purification, the hSDCT2 protein was obtained. Conclusion: hSDCT2 couble be expressed in E.coli system stably.