人7型腺病毒检测细胞系的构建与鉴定

Construction and identification of detection cell line for HAdV7

目的构建人7型腺病毒（human adenovirus type 7，HAdV7）检测细胞系。方法将HAdV7中的Hexon基因克隆至带有绿色荧光蛋白基因的慢病毒表达载体pLV-EGFP（2A）-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稀释法，获得HAdV7感染的检测细胞系HEp-2／GFP。结果细胞系HEp-2／GFP感染HAdV7后24h，可观察到GFP的表达绿色荧光，而作为对照组的HEp-2／GFP细胞感染双链DNA病毒（如EB病毒和乙型肝炎病毒），并未观察到绿色荧光。此检测细胞系用不同滴度的HAdV7感染，至少可以检测到10PFU／mL滴度的HAdV7。结论成功构建了能检测HAdV7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HAdV7感染的诊断方法之一。

Objective To construct detection cell line for human adenovirus type 7. Methods Hexon gene of HAdV7 was cloned into pLV-EGFP （2A） -Puro plasmid with green fluorescent protein gene, and the pLV-EGFP-hexon plasmid was obtained. With the packaging plasmids p-Pax and pMD, pLV- EGFP-hexon plasmid were transfected into 293T ceils by liposome Lip 2000 and lentivirus infected HEp-2 cells were obtained. After pressure screening with puromycin and serial dilution, the detect cell line HEp-2/ GFP for detecting human adenovirus type 7 infection were constructed. Results In 24 hours after HAdV7 infection, the expression of GFP in HEp-2/GFP cells can be observed in the green fluorescence, while the control group of HEp-2/GFP cells infected with double strand DNA virus, such as EBV, HBV and other viruses, showed no green fluorescence. The results of infections on the cell line with different titers of HAdV7 indicated that at least 10 PFU/mL of HAdV7 can be detected. Conclusions The cell line HEp-2/ GFP was successfully constructed and can be used to detect HAdV7 with good speciftcity and sensitivity. The method can be used for diagnosis of HAdV7 infection.