pEGFP-N2-CASK表达质粒的构建及鉴定.doc

  pEGFP-N2-CASK 表达质粒的构建及鉴定 陈鸿1，李原1，朱云霞2，王尧1** （1. 东南大学附属中大医院内分泌科，南京 210009； 5 10 15 20 25 30 2. 南京医科大学江苏省人类功能基因组重点实验室，南京 210029） 摘要：【目的】构建能表达大鼠 CASK 蛋白的质粒。【方法】设计一对用于扩增大鼠 CASK 的编码序列（Coding Sequence，CDS）的引物，以大鼠胰岛β细胞系 INS-1 细胞中提取的总 RNA 逆转录所得的 cDNA 为模板，经过 PCR 扩增目的基因片段，与表达载体 pEGFP-N2 连接后 转化 DH5α感受态细胞，均匀涂布于含卡那青霉素（Kana+）的琼脂平板上，筛选阳性菌落， 提取质粒进行酶切鉴定并送基因公司测序分析。经测序证实插入的片段序列与设计序列完全 一致的重组质粒转染 INS-1 细胞后，用 Western blot 的方法鉴定表达质粒构建是否成功。 【结果】重组质粒经单酶切和测序鉴定证实插入序列完全一致，融合蛋白 pEGFP-N2-CASK 在 INS-1 细胞中成功表达。【结论】成功构建大鼠 CASK 融合蛋白表达质粒 pEGFP-N2-CASK。 关键词：CASK；构建；pEGFP-N2；表达 中图分类号：R587.1 Construction and identification of expression plasmid pEGFP-N2-CASK Chen Hong1, Li Yuan1, Zhu Yunxia2, Wang Yao1 (1. Department of Endocrinology , Zhongda Hospital, Southeast University , NanJing 210009; 2. Key Laboratory of Human Functional Genomics of Jiangsu Province, NanJing 210029) Abstract: Objective To construct the expression plasmid of rat CASK protein. Methods Designed a pair of parimers used to amplify the coding sequence of the rat CASK, the cDNA was reverse transcriptased form total RNA extracted from the rat pancreatic islet beta cells Ins-1 as a template, after amplification the target gene fragment was inserted into the vector of pEGFP-N2, and transfected with competent cells DH5α, the positive clones were identified by agar plate containing kanamycin, and the recombinant plasmids were extracted and confirmed by complete sequencing. Transfect the recombinant plasmids confirmed by sequencing that the inserted fragment and design sequence is exactly the same into INS-1 cells，and identify if the expression plasmid is successful by western blot. Results Recombinant plasmid by single enzyme digestion and sequencing confirmed the insertion sequence is exactly the same, fusion protein pEGFP-N2-CASK was successfully expressed in INS-1 cells. Conclusion The expression plasmid pEGFP-N2-CASK of rat CASK protein is successfully constructed. Keywords: CASK; construct; pEGFP-N2; expression 35 0 引言 研究证实钙 /钙调