关键词: 人细胞周期蛋白D1, 表达, 包涵体, 融合蛋白, 蛋白质纯化, 大肠杆菌

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 a fter IPTG induction and its content was approximately 23% of total bactrerial pr oteins. 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 prote in. 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%.

Key words: human cyclinD1, expression, inclusion body, fusion protein, protein purification, E.coli BL21