利用反向遗传技术制备重组人类偏肺病毒

Generation of Human Metapneumovirus Using Reverse Genetics Technique

摘要:

利用反向遗传技术, 将包含人偏肺病毒2个血清型A和B代表株NL/1/00和NL/1/99全基因组cDNA的质粒及其4种辅助蛋白N、P、L、M2.1的表达质粒pCITE-N、pCITE-L、pCITE-P、pCITE-M2.1分别共转染BSR-T7细胞以制备重组hMPV。3 μg 全长cDNA质粒, 辅助蛋白质粒pCITE-N、pCITE-L、pCITE-P、pCITE-M2.1分别为0.3 μg、0.15 μg、0.3 μg和0.24 μg, 通过Lipofectamine 2000转染BSR-T7细胞,

Abstract:

The full length cDNA clones for hMPV NL/1/00 and NL/1/99 strains from both subtypes, and the four helper vectors pCITE-N, pCITE-L, pCITE-P and pCITE-M2.1 for expressing major viral proteins were co-transfected into BSR-T7 cell to rescue live virons by reverse genetics technique. BSR-T7 cells were transfected using Lipofectamine 2000 with 3 μg of the full-length cDNA plasmid, 0.3 μg of pCITE-N, 0.15 μg of pCITE-L, 0.3 μg of pCITE-P, and 0.24 μg of pCITE-M2.1. Three days after transfection, cells were subjected to one -80°C freeze-thaw cycle to prepare lysates. The lysate was used to inoculate Vero E6 cells. The obvious CPE in Vero E6 cells was observed 6 days after inoculation. The 450 bp RT-PCR products was accordant with the expectant. The specific immunofluorescence was observed for inoculated positive cells. The results demonstrated that infectious hMPV was successfully rescued. The NL/1/00 recombinant virus grew to peak titer of 106.4 TCID50/mL in Vero-E6 cells at 6 days after inoculation and 105. 33 TCID50/mL for recombinant NL/1/99 virus.