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(E.coli)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EcoR Ⅰ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E.coli BL21中,经异丙基硫代β-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.%Objective To construct GST-LMO1 fusion protein expression vector and induce its expression in Escherichia coli(E.coli). Methods The coding sequence of LMOl and its deletion fragments were amplified from the human fetal brain as the template by PCR and inserted into pGEX-5X-2 by BamH I and EcoR I . The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing. Then they were transformed into E.coli BL21, induced by IPTG and identified by SDS -PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-5X-2-LM01 and its deletion mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The GST-LM01 fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid of LMOl and its deletion mutants were successfully constructed and the expression of fusion proteins in Escherichia coli was con-finned. This study provides the basis for the further research on the structure and function of LMOl.
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