人Endostatin基因的克隆及其在hela细胞中的表达

发布时间：2018-01-31 23:58

  本文关键词： Endostatin RT-PCR 基因克隆 脂质体转染 免疫细胞化学　出处：《内蒙古大学》2005年硕士论文　论文类型：学位论文

【摘要】：人内皮抑素(Endostatin)是第一个进入临床研究的内源性血管生成抑制剂,是胶原蛋白ⅩⅧC末端非胶原区NCl结构域的20KDa的片段。它可以抑制内皮细胞的增殖和转移,从而降低肿瘤内血管生成,阻断营养通路,达到杀死肿瘤细胞的目的。其最大的优点为无毒性和无抗药性。本文利用反转录PCR克隆人Endostatin基因,分别构建到分离表达载体pIRES_2-EGFP和融合表达载体pEGFP-C_1中,然后把两个重组表达载体利用脂质体介导法分别转染真核细胞hela,48小时后可在荧光显微镜下观察到被转染细胞发出的绿色荧光,传代10次后,绿色荧光仍持续表达,说明是稳定转染。并且,利用兔抗人Endostatin抗体对已转染的细胞作免疫细胞化学分析,DAB显色后,在显微镜下观察,结果显示,转染的细胞显棕红色,没有转染的细胞不显色。这说明,Endostatin在转染的细胞内稳定遗传与表达。从而为基因药物和基因治疗的研究奠定了基础。
[Abstract]:Human endostatin (Endostatin) is the first to enter the clinical study of the endogenous angiogenesis inhibitor is terminal collagen XVIII C noncollagenous domain NCl domain fragment of 20KDa. The proliferation and it can inhibit endothelial cell migration, thereby reducing tumor angiogenesis, blocking nutritional pathway to kill the tumor cells. Its biggest advantage is non-toxic and without resistance. In this paper, using reverse transcription PCR cloning of human Endostatin gene, were cloned into the expression vector pIRES_2-EGFP separation and fusion expression vector pEGFP-C_1, then the two recombinant expression vector by liposome mediated method were transfected into eukaryotic cells HeLa, after 48 hours can be observed in fluorescence under the microscope, green fluorescence was transfected cells from the 10 passage, the green fluorescence continued to express, that is stable transfection. And anti human Endostatin Using Rabbit anti body to turn Immunocytochemical analysis of stained cells, DAB staining, observed under the microscope showed that the transfected cells were transfected cells without brownish red, no color. This shows that the expression of Endostatin and genetic stability in transfected cells. It laid the foundation for the study of gene therapy for drug and gene.

【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【引证文献】

中国硕士学位论文全文数据库 前1条

1 张明富;禽传染性支气管炎病毒S1基因与猪IgGFc基因的克隆及在Hela细胞中的融合表达[D];华中农业大学;2006年

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本文编号：1480344