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作 者 ：李 波，倪志勇，李晓东，范 玲*

期 刊 ：西北植物学报 2012年 10期 页码：1971~1976

关键词：GhCOMT1；原核表达；蛋白纯化；Western blotting；

Keywords:GhCOMT1, prokaryotic expression, protein purification, Western blotting,

摘 要 ：为进一步研究GhCOMT1基因的功能，构建了原核表达载体pET-28a-GhCOMT1，酶切鉴定并测序后转化到大肠杆菌BL21(DE3)中，在0.2 mmol/L IPTG浓度条件下分别进行不同温度梯度诱导，用蛋白标记亲和层析柱（His Trap^TM HP）对重组蛋白进行纯化，并用SDS-PAGE和Western blotting方法鉴定表达产物。结果表明：16℃诱导12 h、30℃诱导3 h和37℃诱导3 h后融合蛋白均以包涵体的形式表达，其中16℃诱导12 h的蛋白表达量最大；SDS-PAGE检测目的蛋白相对分子量约为39.748 kD；Western blotting分析表明，目的蛋白能与His多克隆抗体起特异性反应。

Abstract:To investigate the function of the GhCOMT1 gene,the GhCOMT1 gene was insert into the pET-28a vector to construct fusion vector pET-28a-GhCOMT1,and transformed into E.coli BL21 (DE3) cells.The fusion protein could be induced by 0.2 mmol/L IPTG treatment for 12 h at 16℃,for 3 h at 30℃ or 37℃,but the recombinant proteins mainly appeared as inclusion bodies.The most high expression quantity was induced at 16℃ for 12 h.Then to dissolved inclusion body,SDS-PAGE analysis indicated that its molecular mass is about 39.748 kD,Western blotting analysis indicated that the His polyclonal antibody could specifically bound to purified His-GhCOMT1 fusion protein.The prokaryotic expression system of pET-28a-GhCOMT1 is successfully constructed.It can be used to the further application study on function of GhCOMT1.

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