长江江豚TRAIL基因的克隆、体外表达及生物学功能分析

Molecular cloning, in vitro expression and bioactivity of TRAIL (TNFSF10) gene from finless porpoises

摘要:

构建可溶性肿瘤坏死因子相关凋亡诱导配体 (TRAIL) 基因的表达体系，研究其蛋白表达产物对肿瘤细胞凋亡的影响，为以后江豚免疫系统的研究奠定基础。通过RT-PCR技术从江豚Neophocaena phoconoides血液总RNA中反转录扩增出肿瘤坏死因子相关凋亡诱导配体 (简称fTRAIL) 的全长cDNA序列，并将fTRAIL的胞外可溶性 (简称fsTRAIL) 片段连接入表达载体pET43.1a中，在大肠杆菌BL21 (DE3) 中表达并纯化，Western blotting对产物Nus-His-fsTRAIL蛋白进行鉴定。体外用MTT法、台盼蓝拒染法及流式细胞术检测Nus-His-fsTRAIL蛋白对Jurkat细胞和HeLa细胞的影响。成功构建了fTRAIL胞外可溶性片段 (简称fsTRAIL) 与pET43.1a组成的表达载体，并获得Nus-His-fsTRAIL蛋白。体外实验表明，Nus-His-fsTRAIL蛋白能够以剂量依赖的方式抑制Jurkat和HeLa细胞的增殖并诱导其凋亡。Nus-His-fsTRAIL表达产物具有对Jurkat和HeLa细胞体外抗肿瘤活性的作用。

关键词:TRAIL，江豚，细胞凋亡，Jurkat细胞，HeLa细胞

Abstract:

To construct soluble TNF related apoptosis inducing ligand (TRAIL) expression system and investigate the effect of the expression product on tumor cell. It may provide valuable information for research into the immune system of the finless porpoise. The full-length cDNA of TRAIL (designated fTRAIL) was cloned from the total RNA of the finless porpoises blood using RT–PCR techniques and then the extracellular soluble fragments of fTRAIL (designated fsTRAIL) was ligated into pET43.1a. Recombinant soluble fTRAIL (pET43.1a-fsTRAIL) fused with Nus-his tag was efficiently expressed in Escherichia coli BL21 (DE3) and the Nus-His-fsTRAIL protein was purified. The expression of Nus-His-fsTRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fsTRAIL protein on Jurkat and HeLa cells were etected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay, TrypanBlue and Flow Cytometry analysis. The expression system pET43.1a-fsTRAIL was constructed and Nus-His-fsTRAIL protein was expressed successfully. In vitro, the Nus-His-fsTRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner. The Nus-His-fsTRAIL protein has anti-tumor activity against Jurkat and HeLa cells in vitro.

Key words:TRAIL, finless porpoises (Neophocaena phoconoides), apoptosis, Jurkat cells, HeLa cells