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-MEG3VI-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. 0-MEG3Vl-12XMS2bs vector expressing non-coding RNA MEG3 VI was also constructed. pcDNA3. 0-Flag-2XMS2 vector and pcDNA3. 0-MEG3Vl-12XMS2bs vector were co-transfected into HEK293 cells. The level of MEG3 VI 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 immunoprecipitation could significantly enrich MEG3V1 RNA. Conclusion We have successfully constructed expression vectors for RNA-protein interaction studies in vivo.
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