两种基于PNP/ Mep-dR 系统的新型融合自杀基因载体的构建及鉴定

 目的 构建两个新型融合自杀基因表达载体并检测其表达。方法 利用DNA重组技术制备两个融合基因PNP-TK和PNP-CD，将二者分别插入真核表达载体pcDNA3．0中，构建两个融合基因表达载体pcDNA3．0／PNP-TK和pcDNA3．0／PNP-CD；经酶切、PCR及测序鉴定各个重组体。结果 成功构建了两个新型融合基因表达载体并在肝癌细胞株HepG2中检测到二者的表达。结论 两个基于PNP／Mep-dR系统的融合自杀基因表达载体是肿瘤基因治疗中杀瘤谱广、杀伤效应强大的理想载体。

Constructions and Identification of two Fusion Gene Vectors Based on PNP/Mep-dR Suicide Gene System

Objective To construct two novel fusion suicide gene expression vectors, and detect their expression in human hepatocellular carcinoma cell line HepG2. Methods Two fusion suicide gene PNPTK and PNP-CD were generated respectively by recombinant DNA techniques. Two fusion genes were inserted into pcDNA 3. 0 vector separately to construct two suicide gene expression vectors, pcDNA 3. 0/ PNP-TK and pcDNA 3. 0/PNP-CD. Two recombinants were analyzed and identified by recombinant enzyme, PCR and sequencing. Then they were transfected into human hepatocellular carcinoma cell line HepG2 by liposome-mediated method. The expressions of two fusion genes were detected in HepG2 cell line. Results Two fusion genes were constructed successfully and the expression of two fusion genes were detected in human hepatocellular carcinoma cell line HepG2. Conclusion Two fusion suicide gene expression vectors based on PNP/Mep-dR system are effective and broadspectrum vectors for human hepatocellular carcinoma gene therapy.