一种新的血管生成抑制因子—TSF的设计及表达  

TSF-a new angiogenesis inhibitory factor and its design and expression

作　　者：沈先荣[1] 吉冬梅[2] 贾福星[1] 胡幼卿[2] 蒋定文[1] 邓新贤[2] 储智勇[1] 王玲[1] 雷呈祥[1] 黄建生[2] 胡忻然[2] 任大明[2] 

机构地区：[1]海军医学研究所,上海200433 [2]复旦大学生命科学院遗传学研究所遗传学国家重点实验室

出　　处：《中华航海医学与高气压医学杂志》2002年第4期239-245,共7页Chinese Journal of Nautical Medicine and Hyperbaric Medicine

基　　金：国家自然科学基金资助 (3 9970 814 )

摘　　要：目的　选择 PF4和 TSP1的高活性片段设计融合基因— TSF,在大肠肝菌表达 ,细胞水平上检测表达产物的生物学活性。方法　 PF4的 C—末端 13肽和 TSP1的 4 2 9～ 4 5 9片断 31肽通过 Gly-Pro- Gly肽桥设计为融合蛋白— TSF。采用 p GEX- 2 T载体在 E.coli JM10 9中表达 TSF蛋白。采用 MTT方法测定 TSF及其相关物 GST- TSF、PF4 (5 8- 70 )、TSP1(42 9～ 4 5 9)等对血管内皮细胞、平滑肌细胞、QGY772 1人肝癌细胞、HL F人肺成纤维细胞等生长的抑制效应。结果　合成的 TSF融合基因 ,克隆到p GEX- 2 T载体 ,转化 E.coli JM10 9,获得了 TSF蛋白的高效表达。建立了 TSF蛋白的纯化复性方法 ,获得了 GST- TSF融合蛋白及 TSF蛋白。TSF、GST- TSF、PF4 (5 8～ 70 )和 TSP1(42 9～ 4 5 9)均特异抑制血管内皮细胞的生长 ,其中 TSF的活性最强 ,活性大小次序为 TSF>GST- TSF>PF4 (5 8～ 70 ) >TSP1(42 9～ 4 5 9)。结论　融合表达蛋白— TSF在细胞水平上能高效特异抑制血管内皮细胞的生长。Objective To detect the inhibitory effect of TSF protein, which was designed from PF4(58～70) and TSP1(429～459) and expressed in E.coli ,on endothelial cells. Methods PF4(58～70) and TSP1(429～459) were ligated with GLy Pro Gly bridge to generat TSF gene. The TSF gene was ligated and cloned into pUC118 plasmid to sequence. Then ,TSF gene from the correct sequence of pUC118/TSF was cloned into a pGEX 2T expression vector to generat recombined plasmid-pGEX 2T/TSF, and transformed into attenuated E.coli JM 109 to construct recombined M109(pGEX 2T/TSF) strain. With the method of MTT assay, the effects of TSF on the proliferation of endothelial cell were detected. Results TSF was expressed highly in inclusion, and the quantity of TSF was 28% in the total of E.coli proteins. The purification procedure was established, and the purified TSF and its fused protein GST TSF were obtained. TSF significantly suppressed capillary EC proliferation in a dose dependent manner. The fuse protein GST TSF, the peptide PF4(58～70) and TSP1(429～459) also inhibited EC proliferation, respectively, but their inhibition rates were not as high as TSF. Conclusions The design of the TSF gene was successful. TSF has significant inhibitory effect on angiogenesis in vitro. TSF with its anti angiogenic activity, should be a useful source of angiogenesis inhibitor.

关 键 词：血小板因子-4 凝血酶敏感蛋白-1 血管生成 基因表达 血管内皮细胞 肿瘤细胞 设计 

分 类 号：R730.5[医药卫生—肿瘤]