携带TAP标签的重组甲型流感病毒的构建与鉴定

Construction and identification of recombinant influenza A virus carrying TAP-tag

摘要:

【目的】将TAP标签构建到WSN病毒基因组上，得到含有TAP标签的重组流感病毒，以便进行后续的病毒追踪。【方法】利用反向遗传学技术，对甲型流感病毒A/WSN/33 (H1N1)的PA片段进行改造来插入TAP (tandem affinity purification)标签序列。通过病毒拯救得到表达外源标签TAP的重组流感病毒WSN PA-TAP，并对拯救出的重组病毒进行生物学鉴定。【结果】成功拯救出重组流感病毒并命名为WSN PA-TAP。重组病毒基因组测序表明重组病毒的序列正确，利用RNA银染技术观察到重组病毒的全基因组片段。重组流感病毒WSN PA-TAP在MDCK细胞上测定生长曲线，发现该重组病毒的复制能力比野生型WSN弱；Western blotting检测到PA-TAP融合蛋白的表达，其分子质量为96 kDa。【结论】成功拯救出能够表达外源标签TAP的重组流感病毒WSN PA-TAP，为筛选与甲型流感病毒聚合酶有关的宿主蛋白的研究提供了新思路，同时也为以甲型流感病毒为载体携带外源基因的探索提供了重要依据。

Abstract:

[Objective] To rescue the recombinant influenza A/WSN/33 (H1N1) virus expressing the tandem affinity purification TAP-tag. [Methods] The PA sequence of influenza A/WSN/33 (H1N1) virus was modified and then inserted into the TAP sequence to construct a recombinant plasmid pHW-PA-TAP-WSN. The recombinant influenza virus WSN PA-TAP expressing exogenous TAP-tag was rescued by reverse genetics and then identified by plaque assay, RNA sliver staining, etc. [Results] The recombinant influenza viruses WSN PA-TAP were successfully rescued. The sequencing results showed that the sequence of the recombinant virus genome was correct, and the eight fragments of the rescued virus were observed by RNA silver staining. The growth curve of WSN PA-TAP was established with MDCK cells. The recombinant WSN PA-TAP had weaker replication ability than the wild-type WSN. Western blotting showed that the PA-TAP fusion protein had a molecular weight of 96 kDa. [Conclusion] The recombinant influenza virus WSN PA-TAP capable of expressing the exogenous TAP-tag was successfully rescued, which provided a new method for the screening of host proteins related to influenza A virus polymerase. Furthermore, the recombinant influenza virus provides a basis for the exploration of influenza A virus carrying foreign genes.