Expression Of Influenza A Virus Nucleoprotein,Preparation Of Anti-NP Antibodies And Its Preliminary Applications

Background: Influenza is a zoonotic respiratory disease caused by influenza virus.The human health and poultry farming will be influenced when Epidemic occurs,also as the development of social economy.Influenza virus is a negative RNA virus,the genome contains 8 genes.Antigen drift is easily happened through gene mutation and gene rearrangement,and lead to the creation of new subtypes.The emerging subtypes lead the existing vaccines failure.The over frequency use of drugs increases the drug resistance.In addition,the characteristics of influenza such as fast spreading,persistence and unpredictability,cause significant difficulties to the prevention and control of influenza.Although the flu is the first desiease to carry out a global surveillance,but each year there are still varying degrees of influenza outbreak in the world.Therefore,it is very important to improve the diagnosis and detection of influenza virus,to take accurate measures directly when the flu occurs.It is benefit to control the flu spread and find specific ways to treat virus,so then we can efficiently prevent and control influenza.Purpose: Predict and analysis the potential epitopes of NP.Express and purify of NP,provide materials for antibody production and identification.Produce and purify of monoclonal antibody against NP,provide materials for detecting influenza virus and doing research for biological characteristic of NP.Methods:(1)The potential B cell epitopes of NP were predicted through the Garnier-Robson,Chou-Fasman,Kyte-Doolittle,Karplus-Schulz,Emini and Jameson-Wolf methods.(2)The NP gene was amplificated by RT-PCR,and was linked into pET-28a(+)prokaryotic expression vector after double enzyme(EcoR I and Sal I).Optimiation conditions of prokaryotic expression plasmid of pET-28a-NP,so that stably express NP fusion protein in E.coli system.Then,the NP-fusion protein was purified with a nickel column affinity chromatography,and identification purification of protein through Western-blot method.(3)The Balb/c mice were immunized with the purified NP and their spleen cells were fused with mouse myeloma cells.After selection,The F1 mice were injected with positive hybridoma cells and produce ascites contains monoclonal antibodies.(4)The purified antibodies are tested by Western blot,enzyme-linked immunosorbent assay(ELISA)to identify the detectional ability to PR8,Memphis and Aichi.Compare double-antibody sandwich ELISA method with direct ELISA to confirm the sensitivity of Memphis.The cell-mediated immunity experiment is using to identify the specificcombination of antibodies to PR8,Memphis,Aichi in MDCK.Through two ways to location NP in MDCK at different time,the one is PR8 infection,the other is pcDNA3.1-NP transfection.Results:(1)According to the prediction analysis of protein structure,speculated that the 5-10,76-80,88-93,124-127,146-152,162-176,203-216,227-231,241-252,291-296,318-327,367-370,396-399,418-422,430-440,452-455,467-472 and 481-487 segments of amino acids of NP have relatively large potential possibility as NP B cell epitopes.(2)Successfully construct of prokaryotic cell expression system pET-28a-NP,induce and expression the NP in E.coli successfully,obtain the high purity of the NP.(3)After immune and cell fusion,get 5 strains of hybridoma cells which can stably produce anti-NP antibodies.After injection,collect 5 strains of monoclonal antibody ascites.Purify ascites and label antibodies with horseradish peroxidase(HRP).All antibodies and their labeled antibodies can specific combine with NP.(4)Preliminarily test the biological characteristics of anti-NP antibody,demonstrating that the antibody is specific and immunological.Anti-NP antibody can specifically combine with NP in PR8,Memphis and Aichi.After PR8 infecting MDCK,we start to detect NP at 8 h mainly in nucleus;with the time past we can observe NP released from nucleus to cytoplasm.The same process is happened in pcDNA3.1-NP transfect MDCK,but the time that start to detect NP is delayed to 24 h.Conclusion: The research analysis of the dominant epitopes of NP.The prokaryotic cell expression system pET-28a-NP which can express NP was constructed.Using the system obtained a pure NP,and hen successfully prepared high potency,specificity of monoclonal antibody against NP through immune Balb/c and cell fusion.After identify and detection,acquired high purity NP and its antibodies can serve as materials to develop a quick diagnostic kit for influenza,or double-antibody sandwich ELISA kit for subtypes of influenza,sublocation of NP in cells and so on.