恒河猴可溶性肿瘤坏死因子相关凋亡诱导配体的基因克隆、重组表达和功能研究  

Molecular Cloning,Recombinant Expression and Functional Characterization of the Soluble Tumor Necrosis Factor-related Apoptosis-inducing Ligand for the Macaca mulatta

作　　者：樊淼淼 贾殿隆 杨浩[1] 万琳[1] 卢晓风[1] 

机构地区：[1]四川大学华西医院卫生部移植工程与移植免疫重点实验室,成都610041

出　　处：《生物医学工程学杂志》2015年第3期605-611,共7页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(81273419)

摘　　要：人肿瘤坏死因子相关凋亡诱导配体(hTRAIL)因对肿瘤细胞有选择性杀伤作用而成为新型抗肿瘤候选药物。预测的恒河猴TRAIL(mmTRAIL)与hTRAIL高度同源,提示其有杀伤人肿瘤细胞的可能性。但目前尚无mmTRAIL蛋白功能的报道。本文首先从恒河猴外周血单个核淋巴细胞中扩增了可溶性mmTRAIL编码基因,然后构建了pQE30-mmTRAIL表达质粒并进行诱导表达。通过Ni-NTA agarose亲和层析从破菌上清液中获得mmTRAIL蛋白。聚丙烯酰胺凝胶电泳和凝胶过滤层析表明,mmTRAIL在溶液中主要以三聚体形式存在。体外条件下,mmTRAIL对结肠癌细胞COLO205显示强烈的杀伤作用,而对正常细胞BEA2b无害,表明其对肿瘤细胞的杀伤有选择性。裸鼠荷瘤模型进一步证实mmTRAIL能够抑制肿瘤生长,具有抗肿瘤作用。这些结果表明,mmTRAIL能够杀伤人肿瘤细胞,可作为新型肿瘤靶向治疗药物进一步深入研究。Human tumor necrosis factor-related apoptosis-inducing ligand（hTRAIL）might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells.The predicted Macaca mulatta TRAIL（mmTRAIL）is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence,suggesting that mmTRAIL might induce apoptosis of human cancer cells.However,the cytotoxicity of mmTRAIL in human cancer cells has not been investigated.In this paper,it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction（RT-PCR）from monkey peripheral blood mononuclear cells（PBMCs）in our laboratory.Subsequently,an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid.After induction by addition of Isopropyl β-D-1-Thiogalactopyranoside（IPTG）, mmTRAIL was expressed.MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography.SDSPAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution.In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole.In addition,antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts.Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts.These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.

关 键 词：肿瘤坏死因子相关凋亡诱导配体 细胞凋亡 肿瘤靶向治疗 

分 类 号：R73-36[医药卫生—肿瘤] Q78[医药卫生—临床医学]