Development And Identification Of Monoclonal Antibodies Specific To HN09-68Virulent Strain

Newcastle disease is caused by Newcastle disease virus, a very serioushazard acute infectious disease of birds, its main feature is caused by damage to thecentral nervous system disorders and digestive system of birds, the main clinicalsymptoms are high fever, difficulty breathing, loss of appetite, diarrhea and showedneurological symptoms. Worldwide, Newcastle is one of the most serious types ofpoultry diseases, our country isolated Newcastle disease virus since1948. for thoseyears,it has caused great harm to China. Therefore, to strengthen the Newcastledisease diagnosis and detection technology development is significant.In Newcastle conventional serological diagnostic methods, they use serumantibody in the HI tests to identify the virus, but the serum have some cross-reactivitywith avian paramyxovirus. Also, there is only one type of Newcastle disease serotype,so HI test can not identify the different pathogenic types of Newcastle disease virus.But monoclonal antibodies are highly specific, monoclonal antibodies can overcomethe cross-reactivity problem in conventional detection, it is now widely used in manytests.In this study, in order to obtain more anti-NDV monoclonal antibodies, we useNDV HN09-68strain as immunogens. Its average death time (MDT) and intracerebralpathogenicity index (ICPI) is53h and1.75, so it is a virulent strain. And harvst thespleen cells of mice, fuse the NS0cells and spleen cells, using whole virus coatedELISA to screening the positive wells. After three times of limiting dilution, wefinally get7stable hybridoma cell lines that secreting anti-NDV monoclonal antibody,named2C9,3B6,3F6,12D4,13A2,13B11,15H7. HI test and ELISA assay resultsshowed: These monoclonal antibodies that specifically bind to NDV09-68strain virus,but not reacting with F48E9virulent and LaSota.For the further analysis of the7strains monoclonal antibodies, we use theprokaryotic expression system to express proteins of NDV HN09-68to identifyspecificity of the monoclonal antibodies. Because HN protein formed the virusenvelope spike on the surface, And it is very important for Pathogenicity, and is animportant for the host protective immune response. Therefore, monoclonal antibodiesagainst HN protein would be very meaningful.Selecting the main antigen region of HN protein, amplificated the gene, and Construct a recombinant vector named pET-32a-HN, then expression thisrecombinant vector in E.coli BL21, the fusion protein is approximately37.1KDa,mainly in the inclusion bodies. The protein was used to coat the ELISA plate afterpurification and renaturation, The ELISA results showed that the monoclonalantibody2C9、3B6、12D4specifically recognized the HN protein. Due to the highestHI titer of the2C9against NDV HN09-68, so2C9was chosed for neutralization testwith NDV HN09-68virus in SPF chicken embryo. Results showed that2C9couldneutralization the virus, so that2C9might be used in the Clinical.