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张建林，邬开朗，张 雪，朱 应，李 雁，赵伟光**，吴建国
( 武汉大学生命科学院病毒学教育部重点实验室，湖北武汉 430072 )
Construction and Expression of Vectors Expressing siRNA
ZHANG Jian-lin，WU Kai-lang，ZHANG Xue，ZHU Ying，Li Yan，ZHAO Wei-guang**，WU Jian-guo
(College of Life Sciences, Wuhan University/Key Laboratory of the Ministry of Education for Virology, Wuhan 430072, China)
Abstract：The fragment of plasmid pCMV-tag-2B excluding Cytomegalovirus(CMV) promoter sequence was obtained by restriction enzyme digestion. A pair of primers was synthesized to amplify a fragment of the H1 promoter according to reference. The fragment of H1 promoter was amplified and inserted into the fragment of plasmid pCMV-tag-2B excluding CMV promoter sequence and plasmid pGEM-11fz, respectively. The recombinant plasmids were named pCH1 and pGH1. A pair of primers, which included small interfering RNA(siRNA)target site for mRNA of EGFP gene, was synthesized and annealed in vitro, and then inserted into the corresponding vectors pCH1 and pGH1. In addition, the plasmid pEGFP-N3 expressing EGFP protein was transfected into liver tumor line Bel-7402 alone, or in combination with the plasmids producing siRNA to degrade mRNA of EGFP gene. The expression level of EGFP protein was viewed under the microscope. The results indicated that the plasmids including siRNA target site for mRNA of EGFP gene produced siRNA molecular and degraded mRNA of EGFP gene. The vector, PCH and pGH, will be used to study prevention of virus in fection.
Key words：siRNA；Expressing vector；Construction；Identification
摘要：本研究用限制性内切酶消化质粒pCMV-tag-2B，除去巨细胞病毒（Cytomegalovirus, CMV）启动子核苷酸序列，剩下的核苷酸序列作为构建表达siRNA（Small interfering RNA，siRNA）载体的前体。依据文献提供的扩增H1 RNA 启动子核苷酸序列的引物序列合成一对引物，以带有H1 RNA 启动子序列的质粒DNA 为模板扩增H1RNA 启动子序列，插入前体，构建SiRNA 的表达载体pCH1。另外将H1 RNA 启动子插入pGEM-11fz 相应位点，构建瞬时表达载体pGH1。依据EGFP 的有效SiRNA 抑制位点，合成两条分别为64bp 的核苷酸链，通过体外退火，形成双链，然后插入已构建的两个表达载体。将这两个载体分别与表达EGFP 蛋白的质粒pEGFP-N3 共
转染Bel-7402 细胞，观察siRNA 对EGFP 的抑制效应。研究结果表明构建的载体有效表达了siRNA，这些载体可以用于与siRNA 相关抗病毒治疗性试验研究。
关键词：siRNA；表达载体；构建；鉴定；病毒治疗

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