GST-LMO1融合蛋白表达载体的构建及其在原核细胞中的表达

 目的构建GST-LMO1 融合蛋白表达载体，并在原核细胞大肠埃希菌（Ecoli ）中诱导表达。方法以人胎脑文库为模板，用PCR方法法扩增出LMO1基因全长及各个截短突变体，通过BamH Ⅰ和EcoRⅠ酶切位点分别将其定向插入pGEX-5X-2载体中，构建原核表达质粒pGEX-5X-2-LMO1及其截短突变体，通过酶切电泳鉴定和DNA 序列测定正确后，转入Ecoli BL21中，经异丙基硫代茁-D 半乳糖苷诱导表达，SDS-PAGE 和Western blot 鉴定。结果酶切电泳及测序结果证明，成功构建了原核表达质粒pGEX-5X-2-LMO1及其截短突变体，并用Western blot 方法证实了GST-LMO1 全长及各个截短突变体融合蛋白的表达。结论成功构建了LMO1 全长及其截短突变体原核表达载体，并证实了其在原核细胞大肠埃希菌中的表达，为LMO1 结构与功能的研究提供了前提基础。

Construction of GST-LMO1 Fusion Protein Expression Vectors and the Expression in Prokaryotic Cell

Objective To construct GST-LMO1 fusion protein expression vector and induce its expression in Escherichia coli（Ecoli ）.Methods The coding sequence LMO1 of and its deletion fragments were amplified from the human fetal brain as the template by PCR and inserted into pGEX-5X-2 by BamHⅠand EcoRⅠ. The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing. Then they were transformed into Ecoli BL21，induced by IPTG and identified by SDS -PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-5X-2-LMO1 and its deletion mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The GST-LMO1 fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid of LMO1 and its deletion mutants were successfully constructed and the expression of fusion proteins in Escherichia coli was con-firmed. This study provides the basis for the further research on the structure and function of LMO1.