An efficient and rapid influenza gene cloning strategy for reverse genetics system.

Influenza reverse genetics plays vital roles in understanding influenza molecular characteristics and vaccine development. However, current influenza reverse genetics heavily depends on restriction enzyme and ligation for gene cloning. The traditional cloning process of influenza eight fragments for virus rescuing generally requires considerable work. To simplify and increase the pace of gene cloning for influenza reverse genetics system, we developed a rapid restriction enzyme-free ExnaseTM II-based in vitro recombination approach for influenza gene cloning. We used this strategy rapidly and successfully to clone influenza eight genes both from viruses PR8 and H9N2 for virus rescuing. Our data demonstrate that the strategy developed here can accelerate the process of influenza gene cloning into reverse genetics system, and shows high potential for applications in both influenza basic and applied research.