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目的:构建携带人环核苷酸门控离子通道基因(HCN4)、发育调控相关转录因子基因(Tbx3)及增强型绿色荧光蛋白基因(EGFP)的慢病毒载体,获得可供转染的滴度,为进一步研究Tbx3对HCN4异位表达及细胞起搏功能的影响提供高效的慢病毒转基因平台。方法:应用聚合酶链反应(PCR)方法,扩增HCN4及Tbx3基因,利用Gateway技术分别构建出HCN4-Tbx3-EGFP、HCN4-EGFP、Tbx3-EGFP、EGFP 4种慢病毒表达载体。经PCR及基因测序鉴定后,脂质体法转染293FT包装细胞,产生相应慢病毒,测定其滴度,荧光显微镜下观察EGFP的表达。结果:PCR和测序证实,构建出携带有HCN4-Tbx3-EGFP、HCN4-EGFP、Tbx3-EGFP及EGFP基因的慢病毒表达载体。分别转染293FT细胞后,荧光显微镜下可见大量绿色荧光,病毒滴度测定结果分别为4.7×107TU/ml、5.2×107TU/ml、4.5×107TU/ml、6.65×107TU/ml。结论:成功构建同时携带HCN4、Tbx3基因及EGFP基因的慢病毒载体并包装出具高效感染力的慢病毒颗粒,为通过HCN4联合Tbx3基因长期稳定的表达来构建窦房结细胞的实验研究提供高效稳定的转基因技术平台。
OBJECTIVE: To construct a lentiviral vector carrying human circular nucleotide-gated ion channel gene (HCN4), developmental regulatory transcription factor (Tbx3) and enhanced green fluorescent protein (EGFP) gene and to obtain transfection titer , In order to further study the impact of Tbx3 on ectopic HCN4 expression and cell pacing function to provide an efficient lentiviral transgene platform. Methods: HCN4 and Tbx3 genes were amplified by polymerase chain reaction (PCR) and four lentiviral expression vectors of HCN4-Tbx3-EGFP, HCN4-EGFP, Tbx3-EGFP and EGFP were constructed by Gateway technology. After identification by PCR and gene sequencing, 293FT packaging cells were transfected with lipofectamine to produce the corresponding lentivirus. The titer of EGFP was determined and the expression of EGFP was observed under a fluorescence microscope. Results: The lentiviral vector containing HCN4-Tbx3-EGFP, HCN4-EGFP, Tbx3-EGFP and EGFP gene was constructed by PCR and sequencing. After transfection with 293FT cells, a large amount of green fluorescence was observed under a fluorescence microscope. The titer of virus was 4.7 × 107TU / ml, 5.2 × 107TU / ml, 4.5 × 107TU / ml and 6.65 × 107TU / ml, respectively. CONCLUSION: Lentiviral vectors carrying both HCN4, Tbx3 and EGFP genes are successfully constructed and packaged with highly infectious lentiviral particles. This study provides an efficient and stable method for the construction of sinoatrial node cells by long-term and stable expression of HCN4 in combination with Tbx3 gene Genetically modified technology platform.