绿色荧光蛋白和胸苷激酶基因共表达的慢病毒载体的构建及表达

Construction of bicistronic lentiviral vectorscarrying HSV-tk and EGFP gene

目的构建人巨细胞病毒广谱启动子（CMV）调控的增强绿色荧光蛋白（EGFP）和自杀基因单纯疱疹病毒胸苷激酶（HSV—tk）共表达的慢病毒载体，获得高效的慢病毒转基因平台。方法采用限制性内切酶酶切、T4DNA连接酶连接等方法，将HSV-tk基因插入慢病毒载体Lenti-ires-EGFP中，构建广谱启动子CMV调控的HSV-tk和EGFP共表达的慢病毒载体（Lenti—CMV-HSV-tk-EGFP）。构建成功后的慢病毒三质粒系统通过磷酸钙沉淀法转染来源于人胚肾细胞系的包装细胞-293T，28h后收集病毒颗粒，测定病毒滴度，并感染人晶状体上皮细胞株、视网膜母细胞瘤细胞株、神经母细胞株、Hela细胞等，荧光显微镜下观察EGFP的表达。RT-PCR检测HSV-tk与EGFP的表达。结果构建的慢病毒载体Lenti-CMV-HSV-tk-EGFP能高效转染各类细胞并持续稳定的表达。RT-PCR的结果表明Lenti-CMV-HSV-tk-EGFP感染细胞能共表达HSV-tk和EGFP，利用该慢病毒载体系统转染人晶状体上皮细胞株时，在复合感染度（MOI）为100时，转染效率接近100％，且传代培养6个月后仍能维持高转染效率。结论成功构建了能转染多种细胞的TK自杀基因和EGFP共表达的慢病毒载体，为眼科自杀基因治疗的实验研究提供高效稳定的转基因技术平台。

Objective To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease. Methods The restriction endonuelease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from peDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site （IRES）-EGFP to construct the bieistronie lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were eo-transfeeted with the lentiviral vector （three plasmids ） by calcium phosphate DNA precipitation. HLEC,HXO-Rb44, SH-SY-SY and Hela cells were transfeeted with viral production and the expression of EGFP was examined under fluorescent microscope after transfeetion. The expression of HSV-tk and EGFP was examined by RT-PCR. Results Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfeetion efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months. Conclusion The bistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.