Bacterial Expression Of Anhui H5N1 Nucleoprotein And Study On The Interaction Of Nucleoprotein And Host Cells

bacterial expression of Anhui H5N1 nucleoprotein and study on the interaction of nucleoprotein and host cellsMaster Candidate:Wang NaifuSupervisor:Zhou Ling,ProfessorCo-mentor:Zhang XiaoguangInstitute for viral disease control and prevention,China CDC,100052 BeijingHuman avian influenza is acute respiratory infectious disease caused by a certain subtypes of avian influenza virus.Under normal circumstances,the avian flu virus does not infect humans,but since 1997 the first human infected by avian flu virus H5N1,there is a succession report that H9N2,H7N7 and H5N1 subtype of avian flu virus cause human infection.Since the end of 2003,highly pathogenic avian flu has been epidemic outbreak and put a great harm on the local economy and people's health in many Asian countries and regions;moreover,it spread across the world to more than 10 countries and regions. From 2003 to date,382 people were infected by avian flu virus H5N1,and the 241 cases dead,of which the majority have occurred in Southeast of Asia.Mainland China also has 30 confirmed cases,of which 20 people were killed(WHO 08 the April 30 release). Recent studies show that:H5 subtype of avian flu virus is more easily than before to get mammals infected,and there is often a high mortality rate owing to lack of immunity in human.The World Health Organization suggests that the disease may be one of the largest potential threats to human's survival.So study on the pathogenic mechanism of avian flu virus is one of the problems to be solved in order to prevent and control of human avian influenza effectively.Nucleoprotein(Nucleoprotein,NP) is an important structural protein of influenza virus:As a specific type protein,NP is the main basis in dividing influenza virus into A,B, C,and it is an important protein in the complex of ribonucleoprotein.Another NP plays an important role in the transcription and replication of virus genome so much as determining the host-specific aspects;in addition NP aslo is the main antigen identified by cytotoxic lymphocytes.During process of infection,NP plays its important role through interacting with many cell proteins:1 the binding of NP to actin F make RNPs retain in the cytoplasm and no longer re-exported into the nucleus 2 as a mediate for the translocation of RNPs by interacting with CRM1 3 NP can interact with BAT1/UAP56 of cells,but function unknown.As NP can play an important role by interacting with many proteins of cell in the infection cycle,so studies on filtrating and determining the unknown cell proteins that may interact with NP have become a hotspot on the pathogenic mechanism of avian influenza virus.In this study pull-down and co-immunoprecipitation technique were used to filtrate and indetify unknown proteins which may interact with NP protein.Firstly,the soluble NP protein of H5N1 with his-tag was expressed in E.coli BL21 by pET30a vector.then the expressed NP was purified by Ni²⁺ chelating chromatography and ionexchange chromatography in order to obtain NP with high purity;Secondly,pull-down and LC-MS/ MS have been used to find the proteins in host cells(BEAS-2B cells)binding to H5N1 NP protein,because will pull-down technology has so many advantages,such as efficient, high-throughput,without markers and dyes etc;collect sufficient amount of proteins captured and identify proteins by database search according to the peptide molecular weight spectrum which from LC-MS/MS analysis.At last,we initial filtrate 20 cell proteins that can interact with H5N1 nucleoprotein.Finally,we selected SFPQ protein from the identitied 20 cell proteins and identified the interaction between SFPQ and NP protein by co-immunoprecipitation technique.Other interaction varify experiments between cell proteins indetified by mass spectrometry with NP protein will be continued.In this study,20 cell proteins were initial filtrated to interact with NP protein by pull-down and LC-MS/MS technique,and the interactions between SFPQ protein and NP protein has been further verified.The finding may contribute to detect laws of interaction between main structure proteins of avian influenza virus H5N1 and host cell proteins,moreover it may provide experimental basis for the pathogenic mechanism of avian flu virus.