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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 identify rat CD47 RNAi adenoviral vector and prepare a recombinant adenovirus with a certain titer of CD47 RNAi gene, which will lay the foundation for the later study on the role and molecular mechanism of CD47 RNAi in chronic heart failure. Methods: According to the CD47 gene sequence of rat, three pairs of small interfering RNA (siRNA) of CD47 gene were designed and synthesized. The complementary oligonucleotide chain was synthesized and the rat CD47 RNAi adenovirus vector was constructed. The target gene was identified . The recombinant adenovirus was co-transfected into human HEK293 cells by lipofectamine 2000 mediated CD47 RNAi adenoviral vector and packaging plasmid for amplification, purification and titer determination. Results: Rat CD47 RNAi adenovirus vector was successfully constructed. After the recombinant adenovirus was amplified and purified, the titer of recombinant adenovirus finally obtained was 1 × 10 ~ (11) PFU / ml. CONCLUSION: The successful construction of a recombinant adenovirus with a certain titer of CD47 RNAi gene provides the basis for the successful study of the role and molecular mechanism of CD47 RNAi in chronic heart failure in rats.