Novel influenza virus A mammalian cell adapted strain and preparation and application thereof
A type of influenza virus, host cell technology, applied in the field of biotechnology and immunology
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Embodiment 1
[0162] Example 1. The NS gene derived from the WSN strain can promote the high-speed growth of the PR8 strain on Vero cells.
[0163] Vero cells are a mammalian cell line recommended by WHO for vaccine production, and their safety has been well proven. However, the backbone virus strain PR8 used in the production of influenza vaccines is an adaptation strain of chicken embryos, and the adaptation of the mammalian cell line Vero The performance is not good enough to meet the requirements of production.
[0164] Therefore, the inventors recombined the PR8 strain with other strains WSN that are more suitable for mammalian cell lines, and improved the adaptability of PR8 on Vero cells. The genes of PR8 strain (except HA and NA) were replaced with WSN by using reverse genetic technology. By comparing the size of the plaques, only the NS gene from WSN replaced the NS gene of PR8. Larger plaques were formed on cells than PR8 ( figure 1 A). And the recombinant virus PR8-WSN M (the ...
Embodiment 2
[0166] Example 2, NS2 K86R The mutation can promote the high-speed growth of PR8 on Vero.
[0167] In order to identify the site of NS gene that promotes the rapid growth of PR8 on Vero cells, the NS gene of PR8 strain was compared with the NS gene of WSN strain. The NS gene of PR8 and the NS gene of WSN shared 11 non-synonymous mutations in the nucleotide sequence, located at the 163,301,308,318,512,537,565,660,661,729,784 nucleotides ( figure 2 , A). Since the NS gene undergoes alternative splicing when it is transcribed into mRNA, two different mRNAs are formed, encoding the nonstructural protein NS1 and the nuclear export protein NS2 (NEP), respectively. These 11 nucleotide mutations resulted in 7 amino acid mutations on the NS1 protein and 6 amino acid mutations on the NS2 protein ( figure 2 , A).
[0168] With the method of reverse genetics, the PR8-based strains with all the above-mentioned NS gene non-synonymous mutations were rescued (103F and 106M are known to st...
Embodiment 3
[0170] Embodiment 3, NS2 K86R The mutation does not change the antigenicity of PR8, as well as the pathogenicity to mice and chicken embryos.
[0171] In mammalian cell culture system, PR8 high-producing strain (PR8-NS2 K86R ) has a faster growth rate than the PR8 wild-type virus, and the inventors need to measure whether its pathogenicity to mammals and birds is also increased accordingly, which is of great significance to the safe production of vaccines.
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