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目的构建针对人BclGL的慢病毒表达载体,检测其对Jurkat T淋巴细胞系凋亡的影响,为进一步研究BclGL在细胞内信号转导及分子功能奠定基础。方法RT-PCR方法扩增人全长BclGL基因,克隆于pGFP-N1质粒中构建BclGL绿色荧光蛋白融合基因(BclGL-GFP),将融合基因克隆于慢病毒载体FUGW并转染293FT细胞包装浓缩慢病毒颗粒。将浓缩慢病毒感染Jurkat细胞,同时设立PBS对照及FUGW病毒阴性对照组,流式细胞术及荧光定量PCR鉴定BclGL在Jurkat细胞中的表达,Annexin V-APC/PI双染检测细胞凋亡变化。结果酶切及测序证实人BclGL慢病毒表达载体构建成功,转染Jurkat细胞72h后Jurkat细胞凋亡明显增加。结论成功构建了高效稳定表达人BclGL的重组慢病毒转染系统,并证实其对Jurkat淋巴细胞的促凋亡效应,为进一步探讨BclGL的生物学功能奠定了基础。
Objective To construct a lentiviral vector expressing human BclGL to detect the effect of BclGL on apoptosis of Jurkat T lymphocyte lines and lay a foundation for further study of BclGL signal transduction and molecular functions. Methods Human BclGL gene was amplified by RT-PCR and cloned into pGFP-N1 plasmid to construct BclGL-GFP. The fusion gene was cloned into lentiviral vector FUGW and transfected into 293FT cells. Virus particles. Jurkat cells were infected with lentivirus, PBS control group and FUGW negative control group were established. The expression of BclGL in Jurkat cells was detected by flow cytometry and fluorescent quantitative PCR. The apoptosis of Jurkat cells was detected by Annexin V-APC / PI double staining. Results The restriction endonuclease digestion and sequencing confirmed that the human BclGL lentiviral vector was constructed successfully. After Jurkat cells were transfected with Jurkat for 72h, the apoptosis of Jurkat cells was significantly increased. Conclusion A recombinant lentiviral transfection system that efficiently and stably expressed human BclGL was successfully constructed and its pro-apoptotic effect on Jurkat lymphocytes was confirmed, which laid the foundation for further study on the biological function of BclGL.