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目的构建锌指抗病毒蛋白(zinc finger antiviral protein,ZAP)基因真核表达质粒,并在人肝癌细胞系HepG2细胞中表达。方法化学合成含第4个CCCH锌指基序的ZAP基因片段,经PCR扩增后,插入pcDNA3.1(+)载体,并在ZAP下游插入报告基因EGFP,构建重组表达质粒pcDNA3.1(+)-ZAP/EGFP。在脂质体Genfectin介导下将重组表达质粒转染HepG2细胞,转染后24 h,荧光显微镜观察EGFP的表达;转染后48 h,RT-PCR法检测转染细胞中ZAP基因和内参GAPDH基因mRNA的转录。结果重组表达质粒pcDNA3.1(+)-ZAP/EGFP经双酶切和测序证实构建正确;转染后24 h,pcDNA3.1(+)-ZAP/EGFP质粒转染的HepG2细胞在荧光显微镜蓝色激发光下可见胞质内有较强的黄绿色荧光;转染后48 h,pcDNA3.1(+)-ZAP/EGFP质粒转染的HepG2细胞可扩增出288 bp的ZAP基因条带和100 bp的内参GAPDH基因条带。结论成功构建了含第4个CCCH锌指基序的ZAP基因真核表达质粒,并在HepG2细胞中获得表达,为下一步深入研究ZAP对肝炎病毒是否具有抗病毒作用以及利用ZAP作为抗病毒治疗的一种新手段奠定了基础。
Objective To construct eukaryotic expression plasmid of zinc finger antiviral protein (ZAP) and express it in HepG2 cell line. Methods ZAP gene fragment containing the fourth CCCH zinc finger motif was chemically synthesized and inserted into pcDNA3.1 (+) vector after PCR amplification. The reporter gene EGFP was inserted downstream of ZAP to construct recombinant plasmid pcDNA3.1 (+ ) -ZAP / EGFP. The recombinant plasmids were transfected into HepG2 cells with Lipofectamine 2000 and transfected into HepG2 cells 24 h after transfection. The expression of EGFP was observed by fluorescence microscope 48 h after transfection. The expression of ZAP gene and GAPDH Transcription of gene mRNA. Results The recombinant plasmid pcDNA3.1 (+) - ZAP / EGFP was confirmed by double enzyme digestion and sequencing. At 24 h after transfection, HepG2 cells transfected with pcDNA3.1 (+) - ZAP / EGFP plasmid were detected by fluorescence microscope blue After 48 h of transfection, HepG2 cells transfected with pcDNA3.1 (+) - ZAP / EGFP plasmid could amplify a 288 bp band of ZAP gene 100 bp internal control GAPDH gene band. Conclusion The ZAP gene eukaryotic expression plasmid containing the 4th CCCH zinc finger motif was successfully constructed and expressed in HepG2 cells. In order to further investigate whether ZAP has antiviral activity against hepatitis B virus and ZAP as antiviral therapy Laid a foundation for a new means.