一种改进型基于荧光蛋白重定位系统的丙型肝炎病毒滴度检测方法的建立

Development of an improved method for determination of hepatitis C virus titer using a fluorescence protein relocalization system

目的建立一种简单、快速检测丙型肝炎病毒(hepatitis C virus,HCV)感染的荧光细胞系检测系统,并用其检测HCV滴度。方法通过RT-PCR法获得干扰素β启动刺激因子-1(interferon beta promoter stimulator-1,IPS-1)蛋白C-端第462-540位氨基酸(C462-540)的基因序列及含有SV40核定位信号的IPS-1(C462-540)基因序列,插入经同样酶切的慢病毒表达载体LV-CAG--EGFP-WPRE、LV-CAG—mCherry(RFP)-WPRE,构建表达EGFP-IPS-1(C462-540)、RFP-NLS-IPS-1(C462-540)的慢病毒重组表达质粒;在此基础上构建含有IPS-1(C462-540)-2A-EGFP-puro、IPS-1(C462-540)-IRES-EGFP-puro的慢病毒重组表达质粒;利用293T细胞包装含有IPS-1(C462-540)蛋白的重组慢病毒;感染Huh7.5细胞,流式细胞仪筛选稳定表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)、红色荧光蛋白(red fluorescent protein,RFP)的Huh7.5细胞,或用puromycin筛选稳定表达EGFP的Huh7.5细胞;利用10倍系列稀释的HCV感染上述细胞,检测HCV的滴度。结果测序表明4种慢病毒表达质粒构建正确;收获的慢病毒感染Huh7.5细胞48 h后,可见EGFP或RFP表达,经流式细胞仪分选获得了稳定表达EGFP-IPS-1(C462-540)、RFP-IPS-1(C462-540)的细胞系,经puromycin筛选获得了稳定表达EGFP-IPS-1(C462-540)的细胞系;经10倍系列稀释的HCV感染后,弥散在细胞质中的点状EGFP、RFP会扩散到整个细胞或定位到细胞核中,在甲基纤维素的限制下形成明显的荧光噬斑点,通过计算荧光噬斑点数得到用Huh7.5-EGFP-IPS-1(462-540)-IRES-PURO、Huh7.5两种细胞检测的HCV滴度分别为(5.6±0.2)×10~5和(5.5±0.2)×10~5 PFU/mL,二者无显著差异。结论成功构建了含有绿色荧光或红色荧光报告系统的细胞系,建立了一种简单、快速检测HCV滴度的新方法。

Objective To develop a simple and rapid fluorescent reporter system for determination of hepatitis C virus （HCV） infection. Methods The amino acid residues 462 - 540 at C-terminus of interferon beta promoter stimulator-1 （IPS-1） gene sequence, IPS-1 （C462-540）-2A-EGFP-puro, and an SV40 nuclear localization signal fused to residues 462 ~ 540 of IPS-1 gene, IPS-1 （C462-540）-IRES-EGFP-puro, were amplified by RT-PCR or PCR, then inserted into lentiviral vectors LV-CAG--EGFP-WPRE and LV-CAG--mCherry（RFP）-WPRE to construct recombinant plasmids for expressions of EGFP-IPS-1 （C462 -540） and RFP-NLS-IPS-1 （C462 - 540）, based on which the recombinant plasmids containing IPS- 1 （ C462-540）-2A-EGFP-puro and IPS- 1 （C462-540）-IRES-EGFP-puro were constructed, respectively. The constructed recombinant lentiviral plasmids containing IPS-1 （C462-540） were packaged in 293T cells. Huh7.5 cells were infected with the obtained recombinant lentiviruses, from which the clones expressing enhanced green fluorescent protein （EGFP） or red fluorescent protein （RFP） were screened by flow cytometry. Ahernatively, the Huh7. 5 cells stably expressing EGFP were screened with puromycin. The cells were infected with 10-fold serially diluted HCV, and determined for virus titer. Results Sequencing proved that four recombinant lentiviral plasmids were constructed correctly. EGFP or RFP were observed in Huh7. 5 cells 48 h after infection with the obtained lentivirus. The cell lines stably expressing EGFP-IPS-1 （C462-540） and RFP-IPS-1 （C462-540） were obtained by screening with flow cytometry, while those stably expressing EGFP-tPS-1 （C462-540） and RFP-IPS-1 by screening with puromycin. After infection with 10-fold serially diluted HCV, EGFP showed diffuse fluorescence in cells while RFP showed nuclear transloeation of fluorescence, which formed obvious fluorescence spots in the presence of methyl cellulose. The HCV titers were obtained by calculating the spot number, which were （5.6 ± 0. 2） x l0s and （5.5 ± 0. 2） ×l0s PFU/mL respectively. Conclusion A fluorescent cell-based reporter system was successfully constructed, based on which a simple and rapid method for dteremination of HCV titer was developed.