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0.2mmol/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 TrapTM 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12h、30℃诱导3h和37℃诱导3h后融合蛋白均以包涵体的形式表达,其中16℃诱导12h的蛋白表达量最大;SDS-PAGE检测目的蛋白相对分子量约为39.748kD;Western blotting分析表明,目的蛋白能与His多克隆抗体起特异性反应。
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.
出处
《西北植物学报》
CAS
CSCD
北大核心
2012年第10期1971-1976,共6页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金-新疆联合基金项目(U1178305)
新疆自治区高技术研究发展计划项目(201111116)
国家自然科学基金项目(31060173)
