Studies Of The Distribution Of Avian Influenza Virus RNA-dependent RNA Polymerase PA,PB1 And PB2 Subunit In Cells Cytoplasm And Preparation Of Monoclonal Antibody

In this study, the segments and full-length PA,PB1 and PB2 fragment of avian influenza virus (AIV) subtype H5N1 were amplified from the cDNA of AIV subtype H5N1 strain A/Duck/Zhejiang/11/00 by RT-PCR and subcloned into pET-32a vector. The nine reconstructed plasmids transformed into in E.coli after sequence analysis. Purified protein which purified by Ni+ column mixed with FCA or FIA to immune rabbit. The prepared polycloned antibody reacted exactally and specifically with the fusion protein by western blot. The full-length PA,PB1 and PB2 gene was inserted into eurokaryotic expression pCAGGS vector and transfected into 293T cells. The anti-serum was inditified by fluorescence. The results showed that the anti-serum could react with nature protein and PA,PB1 and PB2 protein, which have perfect biological activity.To detected the distribution of polymerase PA,PB1 and PB2 subunit in eukaryotic cell, In this study, the full-length PA,PB1 and PB2 fragment of avian influenza virus (AIV) were amplified by RT-PCR and constructed EGFP-tag and nontagged reconstructed plasmid, then transfected into MDCK cell with liposome, and the expression of the green fluoresoent protein and red fluoresoent protein were observed by confocal laser scanning microscopy. The result showed: PA and PB1 subunit were distributed in both the cytoplasm and the nucleus, while the PB2 subunit accumulated in the nucleus, we demonstrate that after the coexpression PB1 and PA ,the fluoresoent enhanced than transfected only PA or PB1.In this experiment, three hybridoma cell strains were developed by confusion SP2/0 cells and spleen cells of immunized mouse with polyclonal antibody. Three positive hybridoma cells strains PB1-1 FGFCB10,PA-1 ADEG9 and PA-2 FAEF1 were determined by ELISA, which ELISA titers of ascites were 106,107 and 105, respectively, the cell supernatants were all103. The subtypes of the three McAbs were all IgG1. The specificity experiment showed: The McAbs could react with H5N1 subtype corresponding antigen, The results sugessed that these McAb would be used for study PA and PB1 of stucture and function.