TAT-凋亡素融合蛋白的表达及其抗肿瘤活性

凋亡素(apoptin)由鸡贫血病毒vp3基因编码，能特异地诱导肿瘤细胞凋亡而对正常细胞 没有毒性,为了获得可转导入细胞内部的凋亡素，将人工合成的编码TAT蛋白转导结构域的DNA片段与凋亡素编码基因克隆入质粒pET-28a内，构建出表达融合蛋白TAT-apoptin的原核表达载体pET-28a-TAT-apoptin.在大肠杆菌Rosetta（DE3）中表达融合蛋白，利用IDA -Ni2+ 亲和柱纯化，葡聚糖凝胶G 25除去尿素后得到可溶的变性蛋白.纯化后的TAT apoptin加入体外培养的人脐静脉内皮细胞(HUVECs)和人肺癌Anip973细胞，对照组加入TAT-麦芽糖结合蛋白（TAT-MBP）. 经免疫组化检测，转导1 h后TAT-MBP分布于以上两种细胞的胞浆和胞核，TAT-apoptin则主要分布于2种细胞的胞浆内，转导24 h后TAT-MBP的亚细胞定位没有变化，TAT-apoptin分别定位于HUVECs的胞浆和Anip973的胞核中.脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）显示转导48 h后，TAT-MBP处理过的 HUVECs和Anip973细胞、TAT-apoptin处理过的HUVECs没有明显改变，而TAT-apoptin处理过的Anip973细胞大量凋亡.以上结果表明TAT apoptin融合蛋白在肿瘤治疗上有潜在的应用价值.

Expression of TAT-apoptin Fusion Protein and Its Anti-tumor Activity

Apoptin, a protein encoded by vp3 gene of chicken anemia virus, can specifically induce apoptosis of tumor cells but has no toxic effect on normal cells. To obtain transducible apoptin, the present study cloned synthesized double-stranded oligomeric nucleotide encoding TAT protein transduction domain and the gene encoding apoptin into the plasmid of pET-28a, thus generating a construct of pET-28a-TAT-apoptin which expressed TAT-apoptin fusion protein. The target protein was yielded in E. coli Rosetta(DE3)and purified by IDA-Ni~ 2 affinity chromatography. Removal of urea via G 25 gel chromatography resulted in soluble denatured protein. Purified TAT-apoptin was then added to cultured human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma Anip973 cells,meanwhile, TAT-MBP was added as negative control. Immuno-histochemistry showed that TAT-MBP entered cytoplasm and nucleus of the above two kinds of cells while TAT-apoptin entered cytoplasm 1 hour after protein transduction. TAT-apoptin migrated from cytoplasm to nucleus in Anip973 24 hours after transduction; in the contrast, there were no changes in subcelluar localization of TAT-apoptin in HUVECs and TAT-MBP in above two kinds of cells. TUNEL assay revealed that massive apoptotic cells could be found in Anip973 cells treated with TAT-apoptin, while no obvious changes occurred to HUVECs treated with TAT-MBP or TAT-apoptin and Anip973 cells treated with TAT-MBP. The results suggest that TAT-apoptin fusion protein may possess potential value in tumor therapy.