绿色荧光蛋白标记重组人偏肺病毒的制备

Preparation of recombinant human metapneumovirus expressing green fluorescent protein

目的人偏肺病毒（human metapneumovirus，hMPV）可致人上、下呼吸道感染。研究利用反向遗传学技术，以带绿色荧光蛋白（GFP）标记的hMPV NL／1／00全长cDNA质粒及4种辅助质粒pCITE-N、pCITE—P、pCITE—M2．1和pCITE—L为基础，在体外制备GFP标记的重组hMPV病毒（命名为rhMPV NL／1／00GFP）。方法采用LipofeetAMINE2000将带GFP标记的hMPV NL／1／00全长cDNA质粒以及蛋白表达质粒pCITE—N、pCITE-P、pCITE—M2．1和pCITE—L共转染BSR—T7细胞，3d后取BSR—T7细胞上清液感染Vero—E6细胞，1～4d后观察Vero-E6细胞出现明显细胞病变（CPE）和绿色荧光，持续观察至第10天。收集病毒上清用于病毒滴度检测。提取培养上清的病毒RNA并通过RT—PCR方法扩增病毒N基因、F基因和G基因验证所获重组病毒。结果Vero—E6细胞接种1～4d后观察到明显CPE和绿色荧光，随后CPE加重，荧光信号加强，持续至感染后10d；第1、5、10、15和20代病毒的滴度波动于10^5.0～10^6.5 TCID50／ml；RT—PCR检测出符合预期大小的910bp（N）、450bp（F）和980bp（G）条带，经上述片段cDNA序列测定和比对表明获得的重组病毒与hMPV NL／1／00原型病毒序列一致。rhMPV NL／1／00GFP重组病毒在体外传代20代后，遗传信号和荧光信号均稳定。结论采用反向遗传学技术成功拯救了具有感染性的重组hMPV病毒，为hMPV感染发病机制及疫苗研究奠定了基础。

Objective To construct the recombinant human metapneumovirus （hMPV） （defined as rhMPV NL/1/00 GFP） in vitro by reverse genetics technique. Methods BSR-T7 cells were transfected using LipofectAMINE 2000 with the full-length cDNA plasmid, and four major protein expressing plasmids, pCITE-N, pCITE-P, pCITE-M2.1 and pCITE-L. After 3 d, cells were subjected to one -80℃ freeze-thaw cycle to prepare lysates. The supernatant of lysate was used to inoculate Vero-E6 cells. After 1-4 d, cells were found for the obvious development of cytopathic effects under light microscope and green fluoroscopic signals under fluorescence microscope, and were observed up to 10 d. The supernatant were collected to detect virus titer. Viral RNA was extracted from the supernatant and reverse transcriptase polymerase chain reaction （RT-PCR） was used to amplify N, F and G genes of rescued virus. Results Cytopathic effects and green fluoroscopic signals was readily and obviously observed after 1-4 d post-inoculation in Vero-E6 cells, then cytopathic effects got worse and green fluoroscopic signals became stronger gradually up to 10 d. The titers of the 1st, 5th, 10th,15th and 20th generation virus ranged from 10^5.0 to 10^6.5 TCID50/ml. Amplicons with size of 910 bp（N） , 450 bp（F） and 980 bp（G） by RT-PCR were accordant with expectant. Nucleotide sequence analysis of above cDNA fragments showed 100% similarity with reported sequence of hMPV NL/1/00 strain. The recombinant virus was genetically constant and GFP-labeled after 20 passages in Vero- E6 cells. Conclusion Recombined hMPV was successfully rescued by reverse genetics technique. This study lays ground for exploring pathogenesis of hMPV infection and development of hMPV attenuated vaccines.