目的: 以腺相关病毒为载体设计和制备同时含有多种DNA病毒基因片段的假病毒,作为阳性对照品用于分子检测领域的人源病毒检测,弥补病毒分子检测方法缺少野生型病毒对照的不足。方法: 采用腺相关病毒载体作为假病毒的骨架,分别将人乳头瘤病毒16型（HPV16）、18型（包括HPV18-1、HPV18-2）和人多瘤病毒HPyV 3种病毒共4个目的基因片段插入到1个病毒载体,通过多质粒共转染的方法进行腺相关病毒假病毒的包装。通过细胞感染实验确认假病毒的感染活性并应用荧光定量PCR法对假病毒基因组进行定量分析。结果: 荧光定量PCR检测,假病毒中HPV16、HPV18-1、HPV18-2和HPyV基因组滴度分别为7.77×108、6.77×108、7.04×108、1.24×109 copies · mL-1。假病毒感染293T/17细胞48 h后,用荧光定量PCR可检测到细胞内假病毒包装的HPV16、HPV18-1、HPV18-2和HPyV的病毒基因序列,其拷贝数分别为1.30×106、6.59×105、6.27×105、4.17×106 copies · mL-1。平行实验制备的报告基因假病毒感染293T/17细胞48 h后,该细胞在荧光显微镜下可观察到明显的绿色荧光,进一步表明该假病毒具有感染活性。假病毒作为阳性对照在人间充质干细胞进行适用性验证,4种病毒基因片段的核酸提取荧光定量PCR回收率分别为94.4%、70.7%、83.1%和90.9%。结论: 本研究腺相关病毒包装方法可以产生具有感染活性的多病毒基因假病毒,该假病毒在干细胞样品病毒荧光定量PCR检查中可作为阳性对照代替多种野生型病毒,不仅降低了阳性对照制备成本,而且更具有生物安全性,在提高检测效率的同时,还能够更好地对qPCR方法从基因提取到基因扩增整个流程进行质量控制。
张峒
,
陈晓菲
,
董莹莹
,
曹译丹
,
王艳辉
,
李慧婷
,
刘明月
,
王新乐
,
崔梦姗
,
付欣悦
,
张瑞瑞
,
庞琳
,
饶春明
. AAV载体-HPV16-HPV18-HPyV假病毒的构建及其在干细胞人源病毒检查中的应用*[J]. 药物分析杂志, 2025
, 45(1)
: 30
-38
.
DOI: 10.16155/j.0254-1793.2024-1301
Objective: To employ adeno-associated viruses as vectors to design and prepare pseudoviruses comprising multiple DNA viral gene fragments. These were used as positive controls for the detection of human viruses in the field of molecular testing, thereby compensating for the absence of wild-type viral controls in viral molecular testing methods. Methods: The adeno-associated viral vectors served as the backbone of the pseudoviruses, with a total of four target gene fragments from three viruses incorporated: human papillomavirus type 16 (HPV16), type 18 (including HPV18-1 and HPV18-2) and human polyomavirus (HPyV) were, respectively, inserted into one viral vector, and the adeno-associated viral pseudoviruses were packaged by multiple plasmid cotransfection. The infectious activity of the pseudoviruses was confirmed by a cell infection assay, and a quantitative analysis of the pseudoviruses genome was conducted using fluorescence quantitative PCR. Results: The titers of HPV16, HPV18-1, HPV18-2, and HPyV genomes in the pseudoviruses were 7.77×108 copies · mL-1, 6.77×108 copies · mL-1, 7.04×108 copies · mL-1, and 1.24×109copies · mL-1, respectively, as determined by fluorescence quantitative PCR. Following a 48 h period of infection in 293T/17 cells, the viral gene sequences of HPV16, HPV18-1, HPV18-2, and HPyV were successfully identified using fluorescence quantitative PCR with the intracellular pseudoviruses. The copy numbers were 1.30×106 copies · mL-1, 6.59×105 copies · mL-1, 6.27×105 copies · mL-1, and 4.17×106 copies · mL-1. Following the infection of 293T/17 cells with the reporter gene pseudoviruses for a period of 48 hours, a distinct green fluorescence was evident under a fluorescence microscope, thereby confirming the infectious activity of the pseudoviruses. The pseudoviruses was employed as a positive control for the verification of applicability in human mesenchymal stem cells, and the recoveries of the four viral gene fragments by fluorescence quantitative PCR for nucleic acid extraction were 94.4%, 70.7%, 83.1% and 90.9%, respectively. Conclusion: The present study demonstrates that the adeno-associated virus packaging method can be employed to produce a multiviral gene pseudoviruses with infectious activity. The pseudoviruses can be employed as a positive control for the replacement of multiple wild-type viruses in the viral fluorescence quantitative polymerase chain reaction (PCR) of stem cell samples.
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