摘要： 为了深入研究GhCOMT3基因的功能,将GhCOMT3基因构建到原核表达载体pET-28a中,并转化到大肠杆菌BL21(DE3)中。用0.2mmol/L IPTG诱导融合蛋白表达,结果发现,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;30℃诱导的蛋白表达量最大,之后对包涵体进行变性溶解,进行SDS-PAGE检测,再经过蛋白标记亲和层析柱(His Trap^TMHP)纯化得到变性的pET-28a-GhCOMT3融合蛋白,用SDS-PAGE和Western blotting检测纯化效果,鉴定表达产物,目的蛋白相对分子量约为39.119kDa,检测结果与预期一致。结果表明,pET-28a-GhCOMT3蛋白在大肠杆菌中获得了高效表达产物,为在蛋白水平上研究GhCOMT3基因功能奠定了基础。

关键词: GhCOMT3, 原核表达, 蛋白纯化, Western blotting

Abstract: To investigate the function of the GhCOMT3 gene,the GhCOMT3 gene was insert into the pET-28a vector to construct fusion vector pET-28a-GhCOMT3,and transformed into E. coli BL21 (DE3) cells. The fusion protein could be induced to express by 0. 2 mmol /L IPTG,in the form of inclusion body at 16℃ for 4 h,30℃ for 3 h or 37℃ for 3 h. The most high expression quantity was induced at 30℃ for 4 h. Then to dissolved inclusion body,the supernatants were analyzed by SDS-PAGE and the results were identified with expecting. Then the protein was purified using His Trap^TMHP to get degenerated protein,SDS-PAGE and Western blotting were then employed to identify target protein,the results were identified with expecting. The prokaryotic expression system of pET-28a- COMT3 is successfully constructed in this experiment. The fusion protein was positive by Western blotting. So we can say that pET-28a-GhCOMT3 protein was expressed in E. coli. It can be used for further investigation on the function of the GhCOMT3 gene in protein level.

Key words: GhCOMT3, Prokaryotic expression, Protein purification, Western blotting

中图分类号: 

• Q78