CD2相关蛋白剪接异构体启动子区及其缺失体的构建和活性分析
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国家自然科学基金(81170661,81300023);江苏省自然科学基金(BK20131020);南京市科技发展基金(201503003)


Construction of plasmid containing promoter of splicing isoform of CD2-associated protein and its deletants and an analysis of their promoter activities
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    目的:构建CD2相关蛋白(CD2-associated protein,CD2AP)剪接异构体启动子区及其缺失体,转染HEK293及HeLa细胞,评价其启动子活性-方法:取稳定生长的HeLa细胞,常规方法提取总RNA,逆转录为cDNA并以此为模板,PCR扩增CD2AP剪接异构体之一CD2AP002转录起始位点上游2 200 bp的启动子区域片段,亚克隆这些片段至pGL-3基本载体上的多个克隆位点,构建含CD2AP002启动子的重组质粒-CD2AP002质粒构建成功后,以此质粒为模板重复上述步骤构建一系列CD2AP002启动子5′侧翼区缺失体-重组质粒及其缺失体转染人胚肾HEK293细胞及人宫颈癌HeLa细胞,双荧光素酶报告基因检测各片段的活性-结果:酶切-核酸序列分析证实,成功构建含CD2AP002启动子的重组质粒及其缺失体-经双荧光素酶活性检测证实不同片段大小的5′侧翼区序列缺失体均具有与片段长度相应的活性-结论:成功构建出在HEK293及HeLa细胞中具有活性的重组质粒及其缺失体-

    Abstract:

    Objective:To construct a luciferase reporter plasmid containing the splicing isoform of CD2-associated protein(CD2AP) human gene promoter and its deletants and to evaluate promoter activity in human embryonic kidney (HEK)-293 cells and HeLa cells. Methods:Extract total RNA from stable HeLa cells and make it to be cDNA by RT-PCR. The 2 200 bp fragment of CD2AP002(one of the spliceosome of CD2AP)was amplified by PCR with cDNA as a template and was directionally cloned into pGL3-Basic multiple cloning sites to construct a new luciferase reporter plasmid. We repeat the above steps to build a series of 5′ deletion promoter plasmids. Transfection of HEK 293 cells and HeLa cells with these new plasmids was performed to induce the relative luciferase activity. Results:DNA sequencing and restriction endonuelease analysis verified the successful construction of the plasmid contaning CD2AP002 human gene promoter and its deletants. Conclusion:The plasmid pGL3-CD2AP002 promoter and its deletants are successfully constructed and have some promoter activities in HEK293 cells and HeLa cells.

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李楠楠,王璐璐,张慧文,金 蕊,周国平. CD2相关蛋白剪接异构体启动子区及其缺失体的构建和活性分析[J].南京医科大学学报(自然科学版),2016,(8):933-936

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  • 收稿日期:2016-02-17
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  • 在线发布日期: 2016-08-19
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