人血管性血友病因子裂解蛋白酶pEGFP-N1真核表达载体的构建及其在HeLa细胞中的表达

发布时间：2018-02-27 05:21

本文关键词： ADAMTS pEGFP-N载体载体构建 HeLa细胞　出处：《中国实验血液学杂志》2013年01期 　论文类型：期刊论文

【摘要】：本研究旨在构建人血管性血友病因子裂解蛋白酶(ADAMTS13)的pEGFP-N1真核表达载体,为进一步研究ADAMTS13在细胞内合成及其分泌提供有力工具。通过PCR方法获取目的基因片段,并在目的基因两端加上限制性酶切位点。限制性内切酶酶切后,连接至pEGPF-N1真核表达载体。连接后获得质粒进行酶切鉴定及DNA测序验证,并转染HeLa细胞。通过荧光显微镜检测绿色荧光蛋白表达,Western blot方法鉴定所得蛋白。结果表明,酶切鉴定及DNA测序确认目的基因与载体正确连接,在荧光显微镜下观察到绿色荧光。Western blot显示,转染后HeLa细胞表达ADAMTS13蛋白。结论:成功构建了ADAMTS13-pEGFP-N1真核表达载体,为进一步研究ADAMTS13合成、分泌及其代谢的生理学机制提供了研究工具。
[Abstract]:The purpose of this study is to construct human von Willebrand factor cleaving protease (ADAMTS13) pEGFP-N1 eukaryotic expression vector, for further study in cellular ADAMTS13 synthesis and secretion. Provide a powerful tool to obtain the target gene fragment by PCR method, and the target gene ends with restriction endonuclease sites. After the connection to the eukaryotic expression vector pEGPF-N1. After the connection of plasmid by enzyme digestion and DNA sequencing, and then transfected into HeLa cells detected by fluorescence microscopy. The expression of green fluorescent protein, Western blot protein. The results show that the method of identification, enzyme digestion and DNA sequencing confirmed the target gene and the vector connected properly, to green fluorescent.Western blot. Under the fluorescence microscope showed that the expression of ADAMTS13 protein in HeLa cells after transfection. Conclusion: the successful construction of the eukaryotic expression vector ADAMTS13-pEGFP-N1 in It is a research tool to study the physiological mechanism of ADAMTS13 synthesis, secretion and metabolism.

【作者单位】：苏州大学附属第一医院、江苏省血液研究所卫生部血栓与止血重点实验室;
【基金】：江苏高校优势学科建设工程资助项目江苏省临床医学中心资助(编号ZX201102) 高等学校博士学科点专项科研基金(编号20103201110011)
【分类号】：R341

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