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

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.

Key words:influenza A virus;tandem affinity purification;reverse genetics