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目的:构建凝血酶敏感蛋白-1N端片段(TSF)的原核表达体系,测定重组蛋白的活性。方法:从人脐静脉内皮细胞cDNA中扩增得到thbs1基因片段,克隆到pET32c(+)载体系统并转化 BL21大肠杆菌,表达纯化得到TSF。MTT法体外测定目的蛋白对ECV304细胞增殖的抑制作用。免疫荧光标记流式细胞仪测定CD36的表达。结果:获得了原核表达的重组TSF蛋白。目的蛋白对CD36阴性的ECV304细胞有增殖抑制作用。结论:低剂量重组TSF蛋白是一个具有临床应用前景的抗肿瘤治疗的辅助方法。
Objective: To construct a prokaryotic expression system of thrombin-sensitive protein-1 (N-terminal) fragment (TSF) and determine its activity. Methods: Thbs1 gene fragment was amplified from cDNA of human umbilical vein endothelial cells, cloned into pET32c (+) vector and transformed into E. coli BL21, and expressed and purified TSF. Inhibition of proliferation of ECV304 cells by MTT assay. CD36 expression was measured by immunofluorescence labeling flow cytometry. Results: The prokaryotic expression of recombinant TSF protein was obtained. The target protein can inhibit proliferation of CD36-negative ECV304 cells. Conclusion: Low-dose recombinant TSF protein is an adjunct to anti-tumor therapy with clinical application prospect.