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

作　　者：裴丽丽[1,2] 章纬菁 卢佳[1] 黄芳[1] 曹倩倩[1] 任文华[1] 

机构地区：[1]南京师范大学生命科学学院江苏省分子医学重点实验室,江苏南京210046 [2]南京医科大学康达学院,江苏连云港222000

出　　处：《生物工程学报》2016年第5期610-620,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31370401)资助~~

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-fs TRAIL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。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 c DNA 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 f TRAIL（designated fs TRAIL） was ligated into p ET43.1a. Recombinant soluble f TRAIL（p ET43.1a-fs TRAIL） fused with Nus-his tag was efficiently expressed in Escherichia coli BL21（DE3） and the Nus-His-fs TRAIL protein was purified. The expression of Nus-His-fs TRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fs TRAIL protein on Jurkat and He La cells were etected by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrasodium bromide（MTT） assay, Trypan Blue and Flow Cytometry analysis. The expression system p ET43.1a-fs TRAIL was constructed and Nus-His-fs TRAIL protein was expressed successfully. In vitro, the Nus-His-fs TRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and He La cells in a dose-dependent manner. The Nus-His-fs TRAIL protein has anti-tumor activity against Jurkat and He La cells in vitro.

关 键 词：TRAIL 江豚 细胞凋亡 JURKAT细胞 HELA细胞 

分 类 号：Q78[生物学—分子生物学]