表达增强型绿色荧光蛋白标记的hBax和hHGF双基因的慢病毒载体构建  

Construction of EGFP Labeled Lentiviral Vector Carrying hBax and hHGF Genes

作　　者：李珂[1] 常青[1] 徐平[1] 高洪波[1] 李贞福[1] 

机构地区：[1]青岛大学医学院附属医院,山东青岛266071

出　　处：《现代生物医学进展》2012年第18期3473-3477,共5页Progress in Modern Biomedicine

基　　金：山东省自然科学基金(2005zrb104001)

摘　　要：目的:构建绿色荧光蛋白标记的hBax和hHGF双基因共表达的重组慢病毒并鉴定。方法:通过重叠PCR技术构建attB1-K-hBAX/T2A/eGFP/P2A/hHGF-attB2基因片段,利用gateway technology构建慢病毒载体质粒pLV.EX2d.null-EF1A>hBAX/T2A/eGFP/P2A/hHGF和阴性对照质粒pLV.EX2d.null-EF1A>eGFP并测序,上述两种质粒分别与辅助质粒共转染293FT细胞包装病毒,荧光显微镜检测病毒滴度。结果:经鉴定慢病毒载体质粒构建正确,荧光显微镜检测hBax和hHGF共表达慢病毒滴度为7.8×107TU/mL,仅表达绿色荧光蛋白的阴性病毒滴度为9×107TU/mL。结论:表达增强型绿色荧光蛋白标记的hBax和hHGF双基因的慢病毒构建成功并获得高滴度的病毒感染液。Objective： To construct and identify lentiviral vector expressing hBax and Hhgf fusion protein labeled with enhanced green fluorescence protein. Methods： The attB1-K-hBAX/T2A/eGFP/P2A/hHGF-attB2 gene fragment was obtained by overlap PCR method. Gateway technology was used to construct the pLV.EX2d.null-EF1A〉hBAX/T2A/eGFP/P2A/hHGF plasmid and pLV.EX2d. null-EF1A〉eGFP plasmid. After sequencing, the lenti virus was packaged through co-transfecting above-mentioned construct into human embryonic kidney cell line-293FT with helper plasmids. Then the virus titer was examined by fluorescence microscope. Results： The re- combinant lentiviral transfer vector plasmids were constructed correctly, the titer of lentiviral-hBAX-eGFP-hHGF was 7.8 ×107 TU/mL, and the titer oflentiviral-eGFP was 9×107 TU/mL. Conclusions： The lentiviral vector was constructed and high titer of lentivirus particles were obtained successfully.

关 键 词：慢病毒载体 肝细胞生长因子 冠状动脉旁路移植术 再狭窄 

分 类 号：Q75[生物学—分子生物学] Q78