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目的构建人腺病毒3型(human adenovirus type 3,HAdV3)检测细胞系。方法将HAdV3中的hexon基因克隆至绿色荧光蛋白EGFP3型基因上,连接慢病毒表达载体(PLV-Puro)上,获得PLV-EGFP-hexon。将其与包装质粒P-Pax、PMD用脂质体Lip2000共转染293T细胞,获得慢病毒颗粒感染HEp-2细胞,经嘌呤霉素压力筛选和系列的释法,获得人腺病毒3型感染的检测细胞系HEp-2/GFP。结果细胞系HEp-2/GFP感染HAdV3后24h,可观察到GFP表达的绿色荧光,而作为对照组的HEp-2/GFP细胞感染双链DNA病毒[如EB病毒(EBV)、乙型肝炎病毒(HBV)等],并未观察到绿色荧光。此检测细胞系用不同滴度的HAdV3感染,至少可以检测到10PFU/ml滴度的HAdV3。结论成功地构建了能检测HAdV3的细胞系HEp-2/GFP,且其检测的特异性和敏感性良好,可以作为检测HAdV3感染的诊断方法之一。
Objective To construct a human adenovirus type 3 (HAdV3) cell line. Methods The hexon gene in HAdV3 was cloned into the green fluorescent protein EGFP3 gene and ligated with the lentiviral vector (PLV-Puro) to obtain PLV-EGFP-hexon. 293T cells were co-transfected with packaging plasmid P-Pax and PMD Liposome Lip2000 to obtain lentivirus-infected HEp-2 cells. Puromycin-pressure screening and serial interpretation were performed to obtain human adenovirus type 3 infection Of the test cell line HEp-2 / GFP. Results GFP-expressing green fluorescence was observed 24 h after infection of HEp-2 / GFP with HAdV3, and HEp-2 / GFP cells as a control were infected with double-stranded DNA viruses such as Epstein-Barr virus (EBV), Hepatitis B virus (HBV), etc.], no green fluorescence was observed. The test cell lines were infected with different titer of HAdV3 and at least 10 HpVU / ml titer of HAdV3 could be detected. Conclusion HEp-2 / GFP, a cell line capable of detecting HAdV3, was successfully constructed and its specificity and sensitivity were good. It can be used as a diagnostic method for detecting HAdV3 infection.