人乳头瘤病毒假病毒体外感染模型的建立及人α防御素5抗病毒作用

目的 建立用人乳头瘤病毒16亚型假病毒(HPV-16 Psv)感染官颈癌细胞模型,并研究3种不同构型人α防御素5(HD-5)抗HPV的作用.方法 将表达HPV-16假病毒衣壳蛋白的重组质粒p16L1L2和含绿色荧光蛋白(GFP)报告基因的质粒Pfwb共转染293FT细胞,分离、纯化病毒颗粒后感染官颈癌细胞株C-33a,同时分别给予20 μg/ml的HD-5/N(天然构型)、HD-5/Acm(半胱氨酸被Acm修饰)或HD-5/Abu(半胱氨酸被Abu替换)处理,培养48 h后荧光显微镜观察和流式细胞分析病毒感染率.结果 Pfwb与P16L1L2成功转染293FT细胞并获得滴度达2.5×108TU/ml的HPV-16假病毒液,镜检和流式细胞术检测显示官颈癌细胞C-33a能够被假病毒感染并表达GFP.HD-5/N、HD-5/Acm和HD-5/Abu对HPV-16感染C-33a细胞的抑制率分别为(96.48±5.67)％、(69.02±7.88)％和(2.71±1.53)％.与HD-5/N相比,HD-5/Acm和HD-5/Abu的抗病毒作用显著降低(P＜0.01).结论 建立了基于流式细胞分析的HPV-16假病毒感染模型并将其用于抗病毒药物活性检测.发现HD-5的空间结构和分子中半胱氨酸对其抗HPV感染的活性有重要作用.

Establishment of a human papillomavirus pseudovirus in vitro infection model and its application to evaluate antiviral activity of human defensin-5

Objective To establish a human papillomavirus16 pseudovirus(HPV16 Psv) infection model in vitro and to determine the anti-HPV effects of human α-defensin 5(HD-5) with different conformations.Methods The 293FT cells were co-transfected with HPV pseudovirus recombinant plasmid P16L1L2 and a GFP reporter plasmid Pfwb.After isolation and purification,the pseudovirus particles were used to infect cervical cancer cell line C-33a.During infection,the C-33a cells were treated respectively with 3 different configurations of HD-5,including HD-5/N with natural configuration,HD-5/Acm with Cys residues blocked by acetamidomethyl(Acm) and HD-5/Abu with Cys residues substituted by α-aminobutyric acid(Abu),at a dose of 20 μg/ml.In 48 h later,the cells were observed by fluorescent microscopy and the viral infection rate was determined by flow cytometry assay.Results The 293FT cells were successfully transfected with Pfwb and P16L1L2,and 2.5×108TU/ml of HPV-16 pseudovirus particles were finally obtained.After culturing,most of C-33a cells were infected by HPV16 pseudovirus,which displayed green fluorescence under fluorescent microscope and could be detected by flow cytometry.The inhibitory rate of HD-5/N,HD-5/Acm and HD-5/Abu on HPV infection was(96.48±5.67)%,(69.02±7.88)% and(2.71±1.53)% respectively.Compared to HD-5/N,antiviral activity of HD-5/Acm and HD-5/Abu was significantly reduced(P<0.01).Conclusion A method for HPV16 pseudovirus infection and anti-HPV analysis based on flow cytometry assay is successfully established.The natural configuration of HD-5 contributes to enhance anti-HPV ability significantly,and Cys residues play important role in the antiviral activity of HD-5.