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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 an expression vector for studying RNA-protein interaction in vivo. Methods The specific binding of MS capsid protein to the MS2binding RNA (MS2bs) was used to construct pcDNA3.0-Flag-2XMS2 and pcDNA3.0-12XMS2bs expression vectors and to express MEG3 non-coding RNA The expression vector pcDNA3.0-MEG3V1-12XMS2bs. Co-transfected cells, RNA immunoprecipitation, the resulting RNA real-time quantitative PCR analysis validation. Results The pcDNA3.0-Flag-2XMS2 and pcDNA3.0-12XMS2bs expression vectors were successfully constructed. Real-time PCR results showed that MEG3V1 non-coding RNA molecules were significantly enriched compared with the control. Conclusion The expression vector for studying RNA-protein interaction in vivo was successfully constructed, which provided a new technical method for RNA-protein interaction research.