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人内皮抑制素在酵母细胞中的表达及其促成纤维细胞有丝分裂活性 |
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孙陆果 姜颖 于永利
摘 要 目的：利用酵母真核细胞表达系统表达出重组人内皮抑制素 (Endostatin)，并对其促成纤维细胞有丝分裂活性进行研究。方法：采 用逆转录-PCR技术自人肝组织获得人内皮抑制素的cDNA，将其克隆入真核表达载体pYEX4T -1，构建含人Endostatin的重组质粒(pYEXEndo)；经全自动序列分析仪测序确证后，将此 重 组质粒转化入酵母细胞(DY150)中进行诱导表达，表达产物经SDS-PAGE分析及Western blot 鉴定。以MTT掺入法初步检测了重组人内皮抑制素对NIH3T3细胞的促有丝分裂活性。结果：经CuSO4诱导的含pYEXEndo的酵母细胞表达出重组人GST-Endostatin 的融合蛋白，此蛋白在凝胶上表现为一约45 kD的阳性区带，在Western blot实验中可被GST 特异性多克隆抗体识别。重组人 GST-Endostatin融合蛋白的粗提物在体外能刺激NIH3T3细 胞增殖。 结论：人GST-Endostatin的融合蛋白在酵母表达系统中高水平表达， 并具有刺激成纤维细胞生长的生物学活性。
关键词 人内皮抑制素 酵母表达系统 促有丝分裂活性 成纤维细胞
中国图书分类号 R392-33
Expression of recombinant human endostatin in yeast and its mitogenic activity on fibroblast cells
SUN Lu-Guo JIANG Ying YU Yong-Li
（Department of Imm un ology,Norman Bethune University of Medical Sciences,Changchun130021）
Abstract Objective：To express recombinant human endostatin in yeast and study its mitogenic activity on 3T3 fibroblast cells.Methods：Human endostatin cDNA isolated from the liver tissue by using RT-PCR was cloned into pYEX4T-1 expression vector. After identifying the sequence of the insert, the recombinant endostatin was expressed in DY150 ye ast cell and the product was identified by Western blot.MTT was used to assay th e activity of recombinant protein.Results：Recombinant hum an GST-Endostatin fusion protein was expressed in DY150 yeast cells under the i nduction of CuSO4 and was observed as a hand of 45 kD on a SDS-PAGE gel and b y We stern blot. The recombinant fusion protein was also found to be able to stimulat e proliferation of NIH3T3 cells. Conclusion：Recombinant h u man GST-Endostatin fusion protein was expressed in the yeast in a high level an d showed mitogenic activity on murine fibroblasts.
Key words Endostatin Yeast expression Mitogenic activity Fibroblast
近年发现的内皮抑制素(Endostatin)是胶原XⅧ的N-末端片段［1］，对血管形成有明显的抑制作用。O'Rei ll y等已克隆表达出重组鼠Endostatin并将其用于荷多种肿瘤的小鼠。Endostatin几乎对所有 肿瘤均表现出强大的抑瘤活性［1］，因此Endostatin有[1] [2] 下一页
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