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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 recombinant lentivirus coexpressed by green fluorescent protein-labeled hBax and hHGF genes. Methods: The attB1-K-hBAX / T2A / eGFP / P2A / hHGF-attB2 gene fragment was constructed by overlapping PCR and lentiviral vector plasmid pLV.EX2d was constructed by gateway technology. Null-EF1A> hBAX / T2A / eGFP / P2A / hHGF And negative control plasmid pLV.EX2d.null-EF1A> eGFP. The two plasmids were cotransfected 293FT cell packaging virus with the helper plasmid, and the virus titer was detected by fluorescence microscopy. Results: The constructed lentiviral plasmid was constructed correctly. The titer of lentivirus coexpressing hBax and hHGF was 7.8 × 107TU / mL by fluorescence microscopy and 9 × 107TU / mL when expressing only green fluorescent protein. Conclusions: The lentivirus expressing the enhanced green fluorescent protein-labeled hBax and hHGF double genes was successfully constructed and a high titer of virus-infected fluid was obtained.