人抑癌基因PTEN的原核表达载体的构建及融合表达

Construction of Prokaryotic Expression Plasmid of the Human Tumor Suppressor Gene PTEN and Its Expression in E.coli

为研究抑癌因子PTEN蛋白的抑癌机理,构建了PTEN cDNA的原核表达载体并进行融合表达。将含有PTEN cDNA的质粒pMD-PTEN经EcoR I和Sal I双酶切,回收PTEN基因片段与经相同酶切的高效原核表达载体pET-44a连接,经序列测定,证实融合型表达载体pET-Nus-PTEN构建成功。转化表达宿主BL21(DE3)后,IPTG诱导表达。经12%SDS-PAGE 凝胶电泳,获得118kD的特异蛋白条带,目的蛋白占细菌总蛋白的17%。结果表明:PTEN基因和Nus基因融合表达成功,获得可溶性Nus-PTEN蛋白。该研究为PTEN蛋白的抑癌机理和基因工程药物的研究打下了基础,这是国内PTEN蛋白在原核细胞中成功表达的首次报道。

To study the function of PTEN protein,the recombinant pET-Nus-PTEN was constructed and expressed in BL21(DE3) E.coli.Positive pMD-PTEN plasmid was digested with EcoR I and Sal I and the PTEN cDNA fragment was ligated to prokaryotic expression vector pET-44a which also been digested with EcoR I and Sal I.Sequence analysis proved that the prokaryotic expression plasmid of PTEN have been constructed successfully.The recombinant plasmid was transformed into BL21(DE3) E.coli.The expression of Nus-PTEN was induced with IPTG,and the expressed product was detected by 12% SDS-PAGE.The target protein was about 118kD and the expression level accounted for 17% of the total cellular protein.The results showed that PTEN protein was expressed fusing with Nus Tag in soluble form.This research has laid foundation for investigating the tumor suppression mechanism of PTEN protein and the probability of genetically engineered medicine of PTEN.This is the first report of PTEN expression in prokaryotic cells in China.