串联MS2衣壳蛋白和MS2特异结合RNA表达载体构建及应用  

Construction and application of multi-MS2 coat protein expression vector and multi-MS2 specific binding RNA expression vector

作　　者：蒋太峰[1,2] 付汉江[1] 刘珊珊[1,3] 金英花[3] 朱捷[1] 李恩民[2] 郑晓飞[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]汕头大学医学院,汕头515041 [3]吉林大学生命科学学院,长春130012

出　　处：《军事医学》2012年第8期595-598,共4页Military Medical Sciences

基　　金：国家重大科学研究计划资助项目(2010CB912801);国家重点基础研究发展计划(973计划)资助项目(2009CB521804);国家自然科学基金资助项目(30873008;30870529);北京市自然科学基金资助项目(5112029)

摘　　要：目的构建体内研究RNA-蛋白相互作用的表达载体。方法利用MS衣壳蛋白可与MS2结合位点(MS2bs)RNA特异性结合的特点,通过设计特异引物,构建pcDNA3.0-Flag-2XMS2和pcDNA3.0-12XMS2bs表达载体,以及表达MEG3非编码RNA的表达载体pcDNA3.0-MEG3V1-12XMS2bs。共转染细胞,进行RNA免疫沉淀,所得RNA进行实时定量PCR分析验证。结果成功构建了pcDNA3.0-Flag-2XMS2和pcDNA3.0-12XMS2bs表达载体,实时定量PCR结果表明,与对照相比能明显富集MEG3V1非编码RNA分子。结论成功构建了体内研究RNA-蛋白质相互作用的表达载体,为RNA-蛋白相互作用研究提供了新的技术方法。Objective To construct two kinds of novel expression vectors for RNA-protein interaction studies in vivo. Methods The pcDNA3.0-Flag-2XMS2 vector containing two repeat MS2 coat protein sequence and the pcDNA3.0- 12XMS2bs vector containing 12 repeat MS2bs（MS2 binding sites） RNA sequence were constructed. The pcDNA3. O- MEG3VI-12XMS2bs vector expressing non-coding RNA MEG3 V1 was also constructed, pcDNA3.0-Flag-2XMS2 vector and pcDNA3.0-MEG3VI-12XMS2bs vector were co-transfected into HEK293 cells. The level of MEG3 V1 RNA from co- transfected cells was detected by RNA immunoprecipitation and real-time PCR. Results pcDNA3.0-Flag-2XMS2 vector and pcDNA3.0-12XMS2bs vector were constructed successfully. Real-time PCR results showed that RNA immunoprecipitati- on could significantly enrich MEG3V1 RNA. Conclusion We have successfully constructed expression vectors for RNA-pro- rein interaction studies in vivo.

关 键 词：MS2衣壳蛋白 RNA-蛋白相互作用 RNA结合蛋白 

分 类 号：R346[医药卫生—基础医学]