大鼠CD47 RNAi腺病毒载体的构建与鉴定

Construction and identification of rat CD47-targetingRNAi adenovirus vector

目的:构建并鉴定大鼠CD47 RNAi腺病毒载体,制备具有一定滴度含CD47 RNAi基因的重组腺病毒,为后期研究CD47 RNAi在慢性心力衰竭中的作用及其分子机制奠定基础。方法:根据大鼠CD47基因序列,设计并合成3对CD47基因的特异性小干扰RNA(siRNA),合成互补的寡核苷酸链,构建大鼠CD47 RNAi腺病毒载体,并对目的基因进行鉴定。通过脂质体2000介导CD47 RNAi腺病毒载体和包装质粒共转染人HEK293细胞,以得到重组腺病毒,对其进行扩增、纯化及滴度测定。结果:成功构建了大鼠CD47 RNAi腺病毒载体。经重组腺病毒扩增与纯化,测得最终获得的重组腺病毒滴度为1×10^(11) PFU/ml。结论:成功构建获得了具有一定滴度含CD47 RNAi基因的重组腺病毒,为后期顺利开展有关CD47 RNAi在大鼠慢性心力衰竭中的作用及分子机制研究提供了研究基础。

Objective：To construct and identification the rat CD47-targetingRNAi adenovirus vector for further studying the role of CD47 RNAi in chronic heart failure and its molecular mechanism.Method：According to rat CD47 gene sequences,three pairs of CD47 gene-specific small nuclear RNA（siRNA）were designed and oligonucleotides complementary were synthesized.The recombinant CD47RNAi adenovirus vectors were transfected into the HEK293 cells with liposome 2000.Then the recombinant CD47RNAi adenovirus vectors were amplified and purified with its titer measured ultimately.Result：We successfully constructed and obtained the rats CD-47 RNAi adenovirus vector with a titer of 1×10^^（11） PFU/ml by amplification and purification.Conclusion：The recombinant rats CD-47 RNAi adenovirus vector can be used in studying the role of CD-47 RNAi in chronic heart failure and its molecular mechanism.