文章摘要
大鼠胰岛素Ⅱ基因启动子克隆与功能验证
Cloning and Functional Verification of Rat Insulin II Gene Promoter
投稿时间:2014-03-21  修订日期:2014-03-31
DOI:
中文关键词: 大鼠  胰岛素Ⅱ基因启动子  功能验证
英文关键词: Rat  Insulin2 gene promoter  Clone  Functional verification
基金项目:国家自然科学(81360135); 广西科学研究与技术开发计划项目(桂科攻1347003-2);广西自然科学(2013GXNSFAA019187)
作者单位E-mail
韦玲静 广西大学 动物科学技术学院 564396867@qq.com 
黄玥萌 广西大学 动物科学技术学院  
严雪瑜 广西大学 动物科学技术学院  
蒋钦杨 广西大学 动物科学技术学院 18876010@qq.com 
兰干球 广西大学 动物科学技术学院  
郭亚芬 广西大学 动物科学技术学院  
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中文摘要:
      【目的】克隆大鼠胰岛素II基因启动子(Rat insulin 2 promoter,RIP 2)序列并对其功能进行验证,为后期生产胰岛特异性表达外源基因的转基因动物打下基础。【方法】以大鼠DNA为模板,PCR扩增RIP 2序列,连接至pMD18-T载体,测序鉴定后提取质粒,用Hind III和BamH I限制性内切酶进行双酶切,回收的RIP2片段插入到不含启动子的pEGFP-1载体,构建含RIP2的真核表达载体pEGFP-1-RIP2,重组质粒转染PK15细胞24h后观察细胞荧光表达情况。【结果】克隆获得RIP2序列(-871至-169位点),长703bp,与参考序列的同源性为99%;包含启动子核心元件TATA盒和CAAT盒;构建重组表达载体pEGFP-1-RIP2,质粒转染细胞能表现出绿色荧光。【结论】获得具有启动子功能的RIP2序列。
英文摘要:
      【Objective】 Cloning Rat insulin 2 promoter (RIP2) and preliminary verified its promoter function, to lay the foundation for producing specific-expression of transgenic animals.【Method】Rat insulin 2 gene promoter were amplified from Wistar rat genome DNA by PCR and connected to the pMD18-T vector, plasmids were used to sequence and double digest by Hind III and BamH I; the RIP2 fragments were inserted into the excluding promoter pEGFP-1 vector, constructing the pEGFP-1-RIP2 expression vector, these constructing plasmids were transfected into PK15cell line, and the cells were observed under the inverted fluorescence microscope after 24 hours. 【Result】 RIP2 sequence is 703bp length (-871~-169 sites), and has 99% homologous with the reference sequence. It contains two core promoter elements TATA box and CAAT box. After transfected pEGFP-1-RIP2 plasmids, the GFP could be observed from PK15 cells.【Conclusion】Functional Rat insulin 2 promoter sequences was cloned.
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