PTEN基因的克隆及其在HepG2细胞中的表达

Molecular cloning of human tumor suppressor gene PTEN and its expression in HepG2 cells

目的克隆人抑癌基因PTEN全长cDNA,构建其真核表达载体并检测其在人肝癌细胞HepG2中的表达。方法采用RT-PCR法从人正常肝组织中扩增PTEN全长cDNA,将之与pMD18-T Simple Vector连接、测序,获得PTEN基因。将该基因与pcDNA3·1(+)载体连接,构建pcDNA3.1-PTEN真核表达载体。用该载体转染HepG2细胞,RT-PCR检测PTEN的表达。结果酶切和测序证实PTEN基因克隆和真核表达载体构建成功。HepG2-PTEN细胞中PTEN mRNA的表达显著高于未转染的HepG2细胞。结论人抑癌基因PTEN在人肝癌细胞系HepG2细胞中能够高效、稳定地表达,为其在肝癌基因治疗研究中的应用奠定了基础。

Purpose To clone human tumor suppressor gene PTEN, to construct its eukaryotic expression vector, and to detect its expression in HepG2 cells. Methods Human PTEN gene fragment was amplified from human hepatic tissue by RT-PCR and cloned into pMD18-T simple vector. After sequencing, the fragment was subcloned into the pcDNA3.1 （ ＋ ） vector and the recombinant eukaryotic expression vector was constructed. PTEN mRNA level of HepG2 cells that transfected with the recombinant vector was evaluated by RT-PCR. Results Restriction enzyme analysis and DNA sequence analysis showed that PTEN gene was cloned and the eukaryotic expression vector was constructed successfully. PTEN mRNA level of HepG2-PTEN cells was obviously higher than that of HepG2 cells which was not transfected. Conclusion Tumor suppressor gene PTEN may be highly expressed in human hepatocellular carcinoma cell line HepG2. It provides basis for further research on targeted gene therapy of human hepatocellular carcinoma.