Generation of Human Immunodeficiency Virus-1 Long Terminal Repeat Reporter Genes by Rapid Polymerase Chain Reaction-Mediated Mutagenesis
Generation of Human Immunodeficiency Virus-1 Long Terminal Repeat Reporter Genes by Rapid Polymerase Chain Reaction-Mediated Mutagenesis
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DOI：
10.1309/lmb66mq8jixdccqj
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发表时间：
2013-08
期刊：
影响因子：
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通讯作者：
Juan Wang;H. Lv;Ling Xu;Jin Yang;Zongxing Yang;N. Wu
中科院分区：
文献类型：
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作者：
Juan Wang;H. Lv;Ling Xu;Jin Yang;Zongxing Yang;N. Wu
目的：人类免疫缺陷病毒（HIV）-1启动子报告系统的开发是研究病毒转录活性的关键。我们设计了一种改进的重叠延伸PCR方法，用于HIV-1长末端重复（LTR）片段的单轮定点突变。方法：采用重叠延伸PCR、Dpn Ⅰ酶切、产物转化等方法。构建了HIV-1 LTR的野生型和系列序列缺失克隆。还测量了基础转录活性。结果：总有效率为53.2%。报告基因分析表明第二个NF-κ B顺式元件及其周围区域在介导HIV-1转录调控中的重要性。结论：该技术适用于病毒与宿主相互作用的高通量功能研究。
Objective: The development of a human immunodeficiency virus (HIV)–1 promoter reporter system is critical for investigating transcription activity of the virus. We designed a modified overlap extension PCR method for single round site-directed mutagenesis of the long terminal repeat (LTR) segment of HIV-1. Methods: The procedure consists of overlap extension PCR, DpnI digestion, and product transformation. Wild-type and serial sequence deletion clones of HIV-1 LTR were constructed. Basal transcription activity was also measured. Results: The overall efficiency for obtaining the desired products was 53.2%. The reporter assay indicated the importance of the second NF-ĸB cis-element and surrounding regions in mediating the regulation of HIV-1 transcription. Conclusion: The technique we investigated is suitable for highthroughput functional studies of virus-host interaction.