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[目的]为研究canstatin对动脉粥样硬化斑块的稳定作用,克隆人canstatin基因,并在COS7细胞中表达出具有 生物活性的canstatin。[方法]利用RT-PCR技术,从人脐静脉血管内皮细胞中扩增canstatin cDNA,并定向克隆入真核表达载 体pSeeTag2C中。用脂质体转染法,将重组质粒pSecTag2C-canstatin转入COS7细胞。培养48 h后,对转化的COS7细胞行PCR 和Western-blot鉴定,用MTT法检测canstatin对人脐静脉血管内皮细胞增殖的抑制作用。[结果]从人脐静脉血管内皮细胞中 扩增出canstatin cDNA片段。测序结果显示,克隆的canstatin cDNA长684 bp,序列与文献报道一致。从pSecTag2C-canstatin转 化的COS7细胞中能特异地扩增出canstatin基因片段,Westem-blot结果显示,转化的COS7细胞中有特异的34 kDa的 canstatin表达。MTT检测显示,表达canstatin的COS7细胞上清能呈剂量依赖性地抑制脐静脉血管内皮细胞的增殖。[结论] 构建了真核表达重组质粒pSecTag2C-canstatin,在COS7中表达出具有生物活性的人canstatin。
[Objective] To investigate the stabilizing effect of canstatin on atherosclerotic plaque, the human canstatin gene was cloned and the bioactive canstatin was expressed in COS7 cells. [Method] The canstatin cDNA was amplified from human umbilical vein endothelial cells by RT-PCR and cloned into the eukaryotic expression vector pSeeTag2C. The recombinant plasmid pSecTag2C-canstatin was transfected into COS7 cells by lipofection. After cultured for 48 h, the transformed COS7 cells were identified by PCR and Western-blot. The inhibitory effect of canstatin on the proliferation of human umbilical vein endothelial cells was detected by MTT assay. [Result] The cDNA fragment of canstatin was amplified from human umbilical vein endothelial cells. The sequencing results showed that the cloned canstatin cDNA was 684 bp in length and its sequence was consistent with that reported in the literature. The canstatin gene fragment was amplified specifically from COS7 cells transformed with pSecTag2C-canstatin. The results of Westem-blot showed that the canstatin expression was specific in the transformed COS7 cells at 34 kDa. MTT assay showed that the supernatant of COS7 cells expressing canstatin inhibited the proliferation of human umbilical vein endothelial cells in a dose-dependent manner. [Conclusion] The eukaryotic expression recombinant plasmid pSecTag2C-canstatin was constructed, and the biologically active human canstatin was expressed in COS7.