蝙蝠TRAIL基因的克隆·表达及生物学功能分析

The Cloning,Expression and Biological Function of the Bat TRAIL Gene

[目的]克隆蝙蝠TRAIL基因,构建蝙蝠可溶性TRAIL原核表达栽体,研究其对肿瘤细胞凋亡的影响。[方法]通过RT-PCR技术克隆蝙蝠TRAIL的全长c DNA序列,实时荧光定量PCR鉴定其组织分布,将其可溶性片段与表达载体p ET43.1a连接,并在大肠杆菌BL21中高效表达和纯化,通过western blotting证实。体外,通过MTT、Trypan Blue和流式细胞术检测纯化的可溶性TRAIL蛋白能否诱导肿瘤细胞的凋亡。[结果]成功克隆蝙蝠TRAIL基因,构建了重组表达载体,获得可溶性TRAIL蛋白。体外试验证明,可溶性蝙蝠TRAIL蛋白能够诱导Jurkat细胞和He La细胞的凋亡。[结论]可溶性蝙蝠TRAIL蛋白可诱导肿瘤细胞凋亡,该研究为蝙蝠免疫系统的研究奠定了基础。

[Objective] To clone gene of the bat TRAIL and construct its soluble prokaryotie expression vector,and investigate its effect on tumor cell apoptosis. [Method]The full-length c DNA of TRAIL from the bat was cloned using RT – PCR techniques. Using real-time PCR to identify its tissue distribution,and its soluble TRAIL gene was linked with p ET43. 1a,efficiently expressed in Escherichia coli BL21（ DE3）and confirmed by Western blot analysis. In vitro,the 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrasodium bromide（ MTT） assay,TrypanBlue and Flow Cytometry analysis the purified soluble TRAIL could induce the apoptosis of tumor cells or not. [Result]The b TRAIL was successfully constructed and obtained a soluble b TRAIL protein. In vitro,the soluble b TRAIL could induce the apoptosis of Jurkat and He La cells. [Conclusion] The purified soluble b TRAIL protein can induce the apoptosis of the tumor cells. These results about b TRAIL gene would provide a basis for understanding the characteristics of the immune system of the bat.