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根据A型流感病毒密码子使用偏嗜性,选取稀有密码子对A/Puerto Rico/8/34(H1N1)病毒NS1基因内部110个氨基酸区域进行密码子同义突变改造,并全基因合成NS基因,利用反向遗传操作技术拯救出含有密码子去优化NS1基因的重组病毒(deoNS)。体外细胞噬斑形成实验和病毒生长曲线证明该病毒在MDCK细胞内的感染和复制能力比野生型病毒低约1000倍;BALB/c小鼠体内致病力实验证明deoNS病毒不能引起小鼠发病和死亡,该病毒在小鼠肺内的复制滴度比野生型病毒低100～1000倍。本研究探索了通过基因组密码子去优化改造途径降低A型流感病毒毒力的可行性,首次证明流感病毒NS1基因密码子去优化同义突变可以降低病毒毒力,为流感减毒活疫苗的研究提供了新的思路。
According to the codon usage bias of influenza A virus, a codon-synonymous mutation in the 110 amino acid region of NS1 gene of A / Puerto Rico / 8/34 (H1N1) virus was selected by using rare codons and the NS gene was all-synthesized , Using reverse genetic manipulation to rescue the recombinant virus (deoNS) containing codons to optimize the NS1 gene. In vitro cell plaque formation assay and virus growth curve demonstrated that the virus in MDCK cells infected and replicated about 1000 times lower than the wild-type virus; pathogenicity experiments in BALB / c mice proved deoNS virus can not cause disease in mice and Died, the virus in mice lung replication titers than wild-type virus 100 to 1000 times lower. In this study, we explored the feasibility of reducing the virulence of influenza A virus through the way of genome codon de-optimization and modification. It is the first time to prove that NS1 gene codon-optimized synonymous mutation of influenza virus can reduce the virulence of virus, which is a live attenuated influenza vaccine Provide a new way of thinking.