绿色荧光标记重组人偏肺病毒空斑形成滴度测定方法的建立

Establishment of plaque assay titration for GFP-labeled recombinant human metapneumovirus

目的 建立用于绿色荧光标记的重组人偏肺病毒（GFP-rhMPV）空斑形成滴度测定方法.方法 基因组中插入绿色荧光蛋白基因的hMPV全序列cDNA质粒和主要蛋白质表达质粒转入细胞293T后获得感染性重组hMPV.GFP-rhMPV在Vero-E6细胞中连续传代提升病毒滴度并保存.将等倍稀释的重组病毒液接种常规制备的Vero-E6细胞单层,用含或不含胰酶的低熔点琼脂精凝胶覆盖细胞,孵育一定时间后,采用荧光显微镜下计数荧光空斑数和抗原抗体蓝斑形成法计算病毒滴度.结果 感染后3 d,荧光显微镜下GFP-rhMPV可在低熔点琼脂糖凝胶覆盖层下形成分界较为清晰的绿色荧光集落,接种后3 d荧光空斑相对独立,便于计数.蓝斑形成法在感染后第5天蓝斑较大,易观察.此前拯救获得的GFP-rhMPV在宿主细胞Vero-E6中的复制滴度可达1×10^6 PFU以上.结论 成功建立了GFP-rhMPV的空斑形成实验滴度定量检测方法,为hMPV的致病机制、防治手段研究奠定了基础.

Objective To establish the plaque assay for the titration of GFP-labeled recombinant human metapneumovims（rhMPV）. Methods Vero-E6 cells were selected as host cells for titration. GFP-labeled hMPV was serially diluted and added to each well to infect the cells. The plates were covered with low melting agarose overlay and incubated for different days in incubator. The plates were then observed under fluorescence microscope for plaques with green flourecence, at the same time, the number of plaques was counted by blue plaque-forming. Results Under the low melting agarose overlay, Vero-E6 cells grew well until the CPE caused by hMPV was seen. Clear green flourescence could be observed the first day post infection, much clearer on the third day post infection but showed somewhat fusion between plaques later on.Blue plaques on the fifth day after infection were large and easy to observe. The recombinant GFP-labeled hMPV could replicate up to 1 × 106 PFU in the Vero-E6 cells. Conclusion Plaque assay for titration of recombinant GFP-rhMPV has been sucessfully established. This methodology will offer a solid base for further studies on pathogenesis and vaccine development of this virus.