目的

本研究将外源新冠病毒刺突蛋白基因（RBD 基因）插入甲型流感病毒（IAV）的减毒活疫苗温敏冷适应病毒株A/Ann Arbor/6/1960（H2N2）聚合酶基因的PA 片段中，并重配H2N2 病毒的PA 包装信号来确定PA 基因的包装信号边界，以便基于边界序列构建可插入外源基因的H2N2 病毒载体。

方法

利用PCR 扩增A/Ann Arbor/6/1960（H2N2）病毒的8 个基因片段，利用反向遗传操作系统，将RBD 基因插入PA 基因，并通过每次增加或减少PA 基因包装信号序列的3 个碱基，重配PA 基因的包装信号。同时，为了完整表达出全长聚合酶基因和外源新冠刺突蛋白基因，在包装信号前依次插入无义突变包装信号序列、猪捷申病毒Ⅰ型2A 多肽序列和编码流感病毒密码子偏好的外源基因序列。利用连接酶将病毒的8 个基因片段与载体进行连接，构建每个基因片段的质粒；通过转染拯救重组病毒，同时利用MDCK 细胞扩增病毒，并在核酸水平鉴定病毒基因的完整性来判断是否破坏流感病毒的包装信号，从而确定流感病毒PA 基因的包装信号边界。

结果

共制备了11 个PA构建体，其编码的包装信号范围为108～138 bp（不包括终止密码子），并在八质粒IAV 拯救系统中成功拯救了包装序列分别为+12 bp +15 bp -6 bp -9 bp -12 bp -15 bp 的重组H2N2 病毒。

结论

本研究成功拯救了6 个重组流感病毒，构建了可插入外源基因的H2N2 载体。H2N2 重组病毒的包装信号区域与PR8 的包装信号区域略有不同，表明不同亚型之间的包装信号位置可不同。

Objective

In this study, the exogenous neocoronin gene (RBD gene) was inserted into the PA fragment of the polymerase gene of the temperature-sensitive cold-adapted virus strain A/Ann Arbor/6/1960(H2N2), a live attenuated vaccine of influenza A virus (IAV), and rewired the PA packaging signals of the H2N2 viruses to determine the boundary of the packaging signals of the PA genes, to construct H2N2 viral vectors that can be inserted into the exogenous genes based on the boundary sequence.

Methods

Using PCR to amplify 8 gene fragments of the A/Ann Arbor/6/1960(H2N2) virus, the RBD gene was inserted into the PA fragment in a reverse genetic manipulation system and the packaging signals of the PA genes were reassembled, that is, the packaging signal sequences of the polymerase genes were added or subtracted by 3 bases at a time. At the same time, in order to fully express the full length polymerase gene and the RBD gene, nonsense mutant packaging signals sequence, Porcine teschovirus-1 2A polypeptide sequence and exogenous gene sequence encoding influenza virus codon preference were inserted before packaging signal. Ligases were used to attach 8 gene fragments of the virus to the vector, and plasmids were constructed for each gene fragment. The recombinant virus was saved by transfection, and the MDCK cell was used to amplify the virus. The integrity of the virus gene was identified at the nucleic acid level to determine whether the packaging signal of influenza virus was damaged and the packaging signal boundary of influenza virus polymerase gene.

Results

A total of 11 PA constructs were created, encoding packaging signals ranging from 108～138 bp (excluding the stop codon). And the recombinant H2N2 viruses with packaging sequences of +12bp +15bp -6bp -9bp -12bp and -15bp were successfully rescued in the eight-plasmid IAV rescue system.

Conclusions

In this study, 6 recombinant influenza viruses are successfully rescued, that is, H2N2 vectors are successfully constructed that can insert exogenous genes. The packaging signal region of the recombinant H2N2 viruses is slightly different from that of PR8, suggesting that the location of packaging signals may differ between different subtypes.