【摘要】 将人细胞周期蛋白D1全长cDNA克隆入原核表达载体pET-28c(+)中,经酶切和测序鉴定正确的重组质粒转化E.coli BL21(DE3)后获得表达菌株.该菌株经IPTG诱导高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白,表达量占菌体总蛋白的23%.包涵体经洗涤和溶解,在变性条件下利用N i2+螯合柱纯化、尿素梯度复性后,得到纯度达98%以上的纯化蛋白.SDS-PAGE显示纯化蛋白的分子量约为43 000,W estern-blot分析表明,在相应分子量处有一特异性条带,说明成功表达和纯化重组人细胞周期蛋白D1.更多还原

【Abstract】 The expression vector pET-28c-cycD was constructed by inserting human cyclinD1 cDNA into pET-28c（+） and was identified by digestion with restriction enzymes and sequence analysis.Then an expression strain was selected after the transformation of the recombined plasmid into E.coli BL21（DE3）,fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction and its content was approximately 23% of total bactrerial proteins.The inclusion body was washed,dissolved and purified by(Ni²⁺) chelate chromatography under denatured condition.The inclusion body protein was renatured by gradual removal of urea through dialysis to obtain the purified fusion protein.SDS-PAGE analysis and Western blotting with an anti-cyclinD1 antibody showed that fusion protein with a molecular weight of about（43 000） was purified and its purity was up to 98%.更多还原