肿瘤坏死因子相关的凋亡诱导配体(TRAIL)cDNA的克隆、表达和活性测定

Cloning, Expression and Bioactivity of TNF-related Apoptosis Inducing Ligand(TRAIL)

肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)能选择性诱导肿瘤细胞凋亡 .为利用基因工程技术获得重组TRAIL蛋白可溶性片段 (sTRAIL) ,设计 1对引物 .利用PCR技术特异性扩增出sTRAIL的cDNA ,克隆于质粒pGEM 3Zf( )的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后克隆于表达质粒pBV2 2 0的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌DH5α .转化菌株经温度诱导 ,SDS PAGE检测和Western印迹鉴定 ,获得重组sTRAIL的高水平非融合表达菌株 .表达量占菌体总蛋白的 2 0 % .对其表达产物进行了初步纯化 ,SDS PAGE结果显示纯度可达 90 %以上 .用L92 9细胞测定其生物学活性表明 。

TNF related apoptosis inducing ligand(TRAIL) can induce specific apoptosis of cancer cells. In order to obtain recombinant TRAIL through genetic engineering, the cDNA coding soluble fragment of TRAIL(sTRAIL) was amplified from human placental cDNA library by PCR method and cloned into pGEM 3Zf(-) vector to analyze the sequence. The result showed that DNA sequence of cloned human sTRAIL was the same as that reported previously. To express sTRAIL,the cDNA was cloned into expression vector pBV220 which was then transformed into E.coli DH5α host bacteria. The recombinant protein was expressed with the yield of 20% total bacterial protein. SDS PAGE and Western blot analysis indicated that the molecular weight of recombinant protein was about 18 5 kD and could react specifically with anti TRAIL monoantibody. The recombinant protein was purified and could significantly induce apoptosis of L929 cells.