从人直肠癌细胞株Colo320中提取总RNA，经RT-PCR扩增出SOD基因片段，经限制性内切酶（BamHI，Sinai）酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上，重组质粒转化大肠杆菌，经菌落PCR和质粒双酶切鉴定、序列测定确认，证实成功地构建了人SOD基因融合表达载体．转化菌经IPTG诱导表达，SDS-PAGE显示有与预期大小约44kD相吻合的融合蛋白带，获得了可溶性表达的目的蛋白，采用Glutathione Sepha—rose4B亲和层析对重组蛋白进行纯化，获得了纯度很高的目的蛋白．%The SOD gene was cloned by RT-PCR from colon carcinoma cell line Colo320. After digested by BamH I and Sma I, it was inserted into the plasmid pGEX-4T-2 with the right reading frame sequence to construct the expression vector, which can express the fusion protein with GST tag. The fusion protein was expressed after inducing with IPTG and purified for antibody preparation by Glutathione Sepharose affinity chromatography.
展开▼
