人腺病毒3型检测细胞系的构建与鉴定

Construction and identification of HAdV3 detection cell line

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 detection cell line. Methods The Hexon gene of HAdV3 was cloned into green fluorescent protein gene, connected to PLV-Puro plasmid, and the PLV-EG FP-hexon plasmid was obtained and then transfected into 293T cells with the packaging plasmid P-Pax and PMD by liposome Lip2000 were transfected into . Lentivirus infected HEp-2 cells were screened after dilution of puromycin and series of pressure screening. Established cell line HEp 2/GFP was tested for detecting human adenovirus type 3 infection. Results The expression of GFP can be observed in HEp-2/GFP cells 24 hours after HAdV3 infection but not in the the control group of HEp-2/GFP cells infected with double strand DNA virus such as EBV, HBV and other viruses. The detection of cell lines with different titers of HAdV3 infec- tions can be low at 10 PFU/ml. Conclusions Cell line HEp 2/GFP was successfully constructed to detect HAdV3 with high specificity and sensitivity.