| 摘要: |
| 目的：克隆表达融合蛋白Tumstatin 183-230-TRAIL，并观察其生物学功能。方法：利用重组PCR技术扩增Tumstatin 183-230-TRAIL(Tu-T)的融合编码序列，构建pMAL-Tu-T融合蛋白表达载体，将其转化大肠杆菌BL21(DE3)，经IPTG诱导表达,得到MBP-Tu-T融合蛋白，用SDS-PAGE鉴定纯化的表达产物。通过体外内皮细胞增殖抑制实验、肿瘤细胞杀伤实验、体外管腔形成抑制试验及电镜检测细胞凋亡情况对其生物学双功能进行鉴定。结果：融合蛋白MBP-Tu-T在BL21中表达率约20%，可明显抑制内皮细胞增殖(IC50 12.5 μg/ml)，杀伤、诱导胰腺癌细胞凋亡并抑制体外管腔形成。结论：融合蛋白MBP-Tu-T具有双功能生物学活性，为进一步研究其靶向性杀伤肿瘤奠定了基础。 |
| 关键词: Tumstatin 183-230 TRAIL 融合蛋白 生物学功能 |
| DOI：10.3724/SP.J.1008.2008.00474 |
| 投稿时间：2008-03-21修订日期：2008-04-21 |
| 基金项目:上海市科学技术发展基金(024319115). |
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| Expression of Tumstatin 183-230-TRAIL fusion protein and identification of its biological functions |
| REN Na,WANG Liang-hua,GAO Yun,SUN Ming-juan,JIAO Yu-liang,JIAO Bing-hua*,GUO Ai-yun |
| (Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China) |
| Abstract: |
| Objective：To express Tumstatin 183-230-TRAIL fusion protein and to observe its biological functions. Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin 183-230 and TNF-related apoptosis-inducing ligand (TRAIL114-281). An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c2; the vector was used to transfect E. coli BL21 (DE3) and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein. The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation, standard tumor cell cytotoxic assay, in vitro tube formation inhibition, and electron microscopic observation (apoptosis). Results: The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation (IC50 12.5 μg/ml ), induced apoptosis of pancreatic cancer cells, and inhibited tube formation. Conclusion: Constructed MBP-Tu-T fusion protein is bifunctional, which lays a solid foundation for further investigation of antitumor effect of Tumstatin 183-230-TRAIL in vivo. |
| Key words: Tumstatin 183-230 TRAIL fusion protein biology activity |
