Interaction Of PB1-F2 Protein Of Influenza A Virus And Human MOAP-1

PB1-F2 which was considered to be one of the important virulence molecule of influenza A virus was discovered by Chen et al.in 2001.A large number of studies had shown that PB1-F2 was pleiotropic in the host cells.The protein could target to mitochondria and induce apoptosis and promote the secondary bacterial infections and enhance the polymerase activities of the virus.The MOAP-1 could interact with Bax in the mitochondria,however when it was over expressed in mammalian cells could trigger cell apoptosis.However,it was still unclear the molecular mechanism about how the interaction between PB1-F2 and the host proteins could trigger the mitochondria apoptosis.Firstly,293T cells were used to extract the total RNA and the MOAP-1 was amplified successfully,then MOAP-1 gene was inserted into the bait carrier pACT2 in this research.Secondly,the pGBKT7-PB1-F2 which stored in the laboratory and the pACT-MOAP-1 that was newly bulit were transformed into yeast cells respectively.Thirdly,the two kinds of yeast liquid were fused in the condition of 24 h 16 ?.Finally,we successfully screened positive clones using defective monoclonal.At the same time,we used yeast two hybrid rotary experiment and galactose enzyme experiment for further verification.In order to corroborate the existence of the interaction between PB1-F2 and MOAP-1,we used GST pull-down,Immuno-coprecipitation and immunofluorescence methods to verify the interaction of PB1-F2 with MOAP-1 in vivo and in vitro.In our study we verified the interaction between PB1-F2 and MOAP-1.To explore the effects of the interaction between PB1-F2 and MOAP-1,the HeLa and 293T cells grown well were plated into the six well plate and were transfected the different eukaryotic expression vector of PB1-F2 and MOAP-1.In order to enhence the stability of them,we supplement the protein inhibitor.In the end,we detected the expression of two protein in the cells using Western blot technology.The results indicated that PB1-F2 could strengthen the stability of the MOAP-1 and enhence the expression of MOAP-1 in cells.The interaction between the PB1-F2 and the host protein MOAP-1 was detected in our research.Moreover the expression of MOAP-1 was enhenced through their interaction.The research provided a basis for the molecular mechanism of apoptosis through the mitochondria-mediated apoptotic pathway that was induced by PB1-F2.It also provided a clue to find the new function of PB1-F2 in host cells.What's more,it made it possible for prevention and treatment of influenza virms and for further researching the pathogenic mechanism of high incidence.