T-bet和c-FLIP双基因共表达质粒的构建及生物学活性检测

Construction of eukaryotic expression vector co-expressing genes encoding T-bet and c-FLIP and its biological function

目的构建携带T-bet和c-FLIP双基因的真核表达载体及检测其生物学活性。方法采用RT-PCR方法从人外周血淋巴细胞的总RNA中扩增出T-bet和c-FLIPcDNA,利用pIRES和pEGFP-C1,构建T-bet和c-FLIP双基因真核表达载体pEGFPT-bet-IRES-c-FLIP。采用非脂质体的脂质型介导载体将pEGFPT-bet-IRES-c-FLIP转染至外周血淋巴细胞,观察T-bet和c-FLIP在外周血淋巴细胞中的表达,并检测其诱导淋巴细胞的抗凋亡作用及Th1/Th2细胞偏移。结果成功构建pEGFPT-bet-IRES-c-FLIP质粒,运用非脂质体的脂质型介导载体将该质粒转染淋巴细胞,其转染率为34.6%。流式细胞仪分析未经pEGFPT-bet-IRES-c-FLIP预处理的淋巴细胞在CH-11作用24h后,细胞的凋亡率分别为(50.12±8.02)%;经pEGFPT-bet-IRES-c-FLIP预处理1d的淋巴细胞,其细胞凋亡率分别下降到(5.73±0.37)%;双基因表达的淋巴细胞的Th1型细胞因子IFN-γ表达水平明显高于空载体表达的淋巴细胞及未经处理的淋巴细胞组(P<0.01),且Th2型细胞因子IL-4表达水平较空载体表达的淋巴细胞及未经处理的淋巴细胞低(P<0.01),差异有统计学意义。结论转染T-bet和c-FLIP共表达基因后的T淋巴细胞可产生抗凋亡作用及向Th1细胞分化。

Objective To construct a eukaryotic expression vector co-expressing genes encoding T-bet and c-FLIP, and to detect its bioactivity in transfected PBMCs. Methods c-FLIP and T-bet genes were cloned from total RNA of lymphocyte with RT-PCR methods, then constructed into the eukaryotic expression vector by using pIRES and pEGFP-C1 vectors. Lymphocyte from blood donated by hepatocellular carcinoma patients infected by pEGFPT-bet-IRES-c-FLIP with QuikFusin Transfection Kit induced the Th1/Th2 shift and produced anti-apoptosis derived from CH-11 antibody, whose anti-apoptosis evaluated with PI stain flow cytometry （FCM）. Results T-bet and c-FLIP genes were successfully cloned from lymphocytes and incorporated into the eukaryotic expression vector. Exposed to CH-11 for 24 h, the apoptosis of lymphocyte preconditioned by pEGF- PF-bet-IRES-e-FLIP for 24 h was significantly decreased, compared with no-preconditioned cells （5.73%±0.37% vs 50.12%± 8.02% ）, indicating that pEGFPT-bet-IRES-c-FLIP preconditioning protected lymphocyte against apoptosis induced by CH-11. Lymphocyte infected by pEGFPT-bet-IRES-c-FLIP significantly enhanced IFN-γ levels and decreased IL-4 levels, compared with no-preconditioned cells and the cells infected by empty vector. Conclusion A eukaryotic expression vector co-expressing genes encoding T-bet and c-FLIP may induce anti-apoptotic activities of lymphocyte and Thl differentiation.