H7N9甲型流感病毒Vero细胞高产疫苗候选株的构建

Construction of a Vero cell-based high-yield H7N9 influenza vaccine candidate strain

目的构建1株基于Vero细胞高产的H7N9亚型流感病毒疫苗候选株，为应对人感染H7N9病毒提供细胞介质的疫苗候选株。方法利用流感病毒反向遗传学技术，将H7N9亚型流行株的HA和NA基因与高产疫苗骨架株PR8-4mut的6条编码病毒内部蛋白的基因，通过6：2重组法拯救出4mut-H7N9病毒。通过病毒学方法检测病毒在Vero细胞上的生长性状和病毒蛋白产量。结果4rout-H7N9病毒在Vero细胞上生产速度更快，可收获更多的病毒蛋白。同时，4mut-H7N9病毒没有改变胰酶依赖性，并保持在小鼠中的低致病性，保证了该高产疫苗候选株的安全性。结论通过6：2重组法，以反向遗传学技术构建的4mut-H7N9疫苗候选株能够极大地提高病毒在Vero细胞中的增殖速度，可作为以Vero细胞为生产介质的H7N9高产疫苗备选株。

Objective To construct an H7N9 vaccine strain by using a previously obtained Verc cell-based high-yield influenza A virus as the donor strain. Methods The recombinant virus strains, 4mut- H7N9 and PR8-H7N9, were respectively rescued with reverse genetics technique by combining the genes en- coding hemagglutinin （HA） and neuraminidase （NA） of H7N9 virus with the 6 internal genes of PR8-4mul or PR8 virus strains. The growth feature of 4mut-HTN9 virus strain was compared with that of PR8-H7N9 vi- rus strain with growth curves and plaque assays. The viral proteins produced by 4mut-HTN9 and PRg-HTNg virus strains were measured by Western blot and Coomassie blue staining. Results The PR8-H7N9 ant 4mut-H7N9 virus strains were successfully rescued. The virus titer of 4mut-H7N9 strain was about 3000 times higher than that of PR8-H7N9 strain at 72 hours after infecting Vero cells. The 4mut-H7N9 virus strair formed plaques of about 1 mm in diameter on Vero cells, while the PR8-H7N9 virus strain only formed pin- point plaques on Vero cells. The levels of viral proteins encoded by purified 4mut-H7N9 virus strain were significantly higher than that of the PR8-H7N9 virus strain as indicated by both Western blot and Coomassie blue staining. Moreover, the 4mut-H7N9 virus strain was less pathogenic than PR8-H7N9 strain in mice, and retained the trypsin dependence for infecting cells. Conclusion The reassortant 4mut-H7N9 vaccine strain as established by reverse genetics technique grew faster and better in Veto cells, suggesting the possi- bility of using it as a candidate vaccine strain whenever facing a potential epidemic of H7N9 virus.