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Expression and Purification of Avian Influenza Virus RNA Polymerase from Eukaryotic Cell and Preliminary Research of the Host Factors Combining with It

Author: LiYin
Tutor: DengGuoHua
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Avian flu PB2 Co-immunoprecipitation Tandem affinity purification Host -related factors
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 103
Quote: 0
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Abstract


In the present study , we take advantage of the the prokaryotic expression influenza virus RNA polymerase basic protein 2 ( polymerase basic protein 2 , PB2) immune BABL / c mice , after cell fusion and hybridoma screening prepared for the influenza virus polymerase subunit PB2 monoclonal antibody 5G11 . The experiments show that 5G11 H5 subtype different strains PB2 protein response . Identified by the monoclonal antibody subtypes , the 5G11 heavy chain IgG2a, the light chain is κ chain . This strain monoclonal antibody from the supernatant of the lysis of virus-infected cells , immune coprecipitation virus RNA polymerase complex . By two-dimensional electrophoresis distinguish coimmunoprecipitation get protein mixture found and identified by mass spectrometry may be related to the viral RNA polymerase combination of host proteins ; addition, we constructed three subunits of the influenza virus polymerase with TAP tag eukaryotic expression of plasmid : pcDNA- 35PB1 ③ The pCDNA - 35PB2TAP and pCDNA - 35PA . These plasmid transfected 293T cells , influenza virus polymerase complex after tandem affinity purification methods . This test successfully established eukaryotic expression and purification of influenza virus RNA polymerase has been expressed in eukaryotic cells influenza virus RNA polymerase complex , a preliminary study of the influenza virus polymerase relative role of host protein as interaction study between the two viral pathogenic mechanism lays the foundation conducive to further our understanding of the pathogenic mechanism of influenza virus , but also conducive to the development of new antiviral drugs for influenza .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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