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

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.