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目的重组并包装干扰素调节因子3(interferon regulator factor 3,IRF3)短发夹RNA(short hairpin RNA,sh RNA)腺病毒,研究该病毒对RAW264.7细胞IRF3基因表达及对细胞增殖活性和吞噬功能的影响。方法采用酶切和连接方法构建p Adeno-U6-CMV-EGFP-IRF3 sh RNA质粒;借助Ad MaxTM病毒包装系统完成IRF3基因干扰腺病毒的重组与包装;IRF3基因表达分别采用real-time PCR和Western blot分析方法检测;RAW264.7细胞的增殖活性和吞噬功能分别采用CCK8分析和吞墨试验检测。结果 IRF3基因的干扰片段及对照序列被正确插入p Adeno-U6-CMV-EGFP质粒AgeⅠ和Eco RⅠ酶切位点之间,经PCR扩增及DNA测序证实;RAW264.7细胞组成性表达IRF3基因,针对IRF3基因后部序列的干扰腺病毒Y2147明显抑制了IRF3 m RNA和蛋白质的表达,而针对IRF3基因前部及中部序列及对照序的干扰腺病毒对IRF3的表达无明显沉默或封闭效应;腺病毒Y2147对RAW264.7细胞的增殖活性及吞噬功能无明显不良影响。结论成功构建并包装了IRF3 sh RNA腺病毒,该病毒能有效抑制RAW264.7细胞IRF3 m RNA和蛋白表达,且对细胞的增殖活性和吞噬能力无不良影响。
Objective To recombine and package IRF3 short hairpin RNA (sh RNA) adenovirus to study the expression of IRF3 in RAW264.7 cells and its effect on phagocytosis and phagocytosis Effect of function. Methods Plasmid pAdeno-U6-CMV-EGFP-IRF3 was constructed by restriction enzyme digestion and ligation. IRF3 gene was subcloned into recombinant adenovirus by means of Ad MaxTM packaging system. The expression of IRF3 gene was detected by real-time PCR and Western blot assay; the proliferation activity and phagocytosis of RAW264.7 cells were detected by CCK8 assay and ink swab test respectively. Results The IRF3 gene fragment was inserted into the plasmid pAdeno-U6-CMV-EGFP between the EcoRI and EcoRI restriction sites and confirmed by PCR and DNA sequencing. RAW264.7 cells constitutively expressed IRF3 gene , The IRF3 mRNA rearranged adenovirus Y2147 significantly inhibited the expression of IRF3 mRNA and protein. However, there was no significant silencing or blocking effect on the IRF3 expression by the interfering adenovirus against the front and middle IRF3 sequences and the control sequence. The adenovirus Y2147 had no obvious adverse effect on the proliferation activity and phagocytosis of RAW264.7 cells. Conclusion The IRF3 sh RNA adenovirus was successfully constructed and packaged. The virus can effectively inhibit IRF3 mRNA and protein expression in RAW264.7 cells, and has no adverse effect on cell proliferation activity and phagocytosis.