Construction Of Luciferase Reporter Gene From AIV Mini Gene And Application In RNPs Function

H5N1 highly pathogenic avian influenza is responsible for one of the most devastating diseases in domestic poultries. H5N1 avian influenza virus caused several infected humans in South Asia suggested this subtype viruses pose severe threat for pubic health. However, the mechanisms for H5N1 avian influenza virus to cross the avian-mammalian host barrier are still not clear..Using an 8-plismid reverse genetics system, Zejun Li discovered that the PB2 gene of the DKGX/22 virus dramatically attenuated the DKGX/35 virus (MLD50, 6.3log10EID50), but the point is in which step does the attenation happens .We constructed RNPs in 293T cell by transfection with pBD-PB1, pBD-PB2, pBD-PA, pBD-NP. The constructed-vector pPolI - Luci-T were co-transfected 293 T cells. The fluorescent light was tested through fluorescence detector test kit and the result showed that functional protease was assembled.Single gene was replaced in RNPs. We constructed recombinant virus polymerase complex with 3 genes from DKGX/35/01 RNPs and 1 gene from DKGX/22/01 RNPs, and 3 genes from DKGX/22/01 RNPs and 1 gene from DKGX/35/01 RNPs. The fluorescence was measures by fluorescence detector, the result showed PB2 gene plays a key rule in determing the activity of RNPs . The test of 2 genes re-assort RNPs activity also confirmed the conclusion. Polymerase activity was significantly reduced through the replacement PB2 protein containing mutation in 699 and 701 Aa, but to a greater impact on 701 points.We can conclude that: the activity of protease was on the line with the the ability of influenza virus pathogencity in mammals. From the Genetic law, DNA-protein - protein function - biological activity: the PB2 gene of influenza virus affects the polymerase complex functions, thereby the activity of RNPs affects the pathogenicity of the influenza virus to mammals markedly.