Establishment Of Monoclonal Antibodies Against Newcastle Disease Virus Matrix Protein And Its Nuclear Localization

Newcastle disease virus(NDV)is a poultry virus that has threatened the poultry industry for a long time.The genome encodes six structural proteins.The Matrix protein is a non-glycoprotein,which is involved in the formation of the membrane of NDV virion.The M protein is located in the innerface surface of viral membrane,the tail of which is adjacent to the nucleocapsid and together constitute the skeleton of the viral membrane.NDV M protein plays a role in virus assembly,budding and release.It has been reported that NDV M protein showed nuclear import after transfection of plamids,but the real nuclear localization of M protein at NDV infection was not confirmed,and the biological significance of M protein nuclear localization is not clear.1.Bioinformatics analysis of M protein of Newcastle disease virus:In this study,the M protein of Newcastle disease virus was analyzed bioinformatics.Including B cell antigen epitope,T cell epitope,secondary structure,3D surface display,etc.,Amino acids at positions 42?56 of M protein were selected as antigen synthesis sequences.2.Preparation of Newcastle Disease Virus M protein Monoclonal antibody:After epitopes analysis,We selected the oligopeptide sequence of M protein for artificial synthesis.After coupling KLH and emulsifying with Freund's complete adjuvant,BALB/c mice were immunized by subcutaneous multi-pointed injection at 50p.g/one.Second and third immunizations were performed with Freund's incomplete adjuvant emulsifying antigen at a two-week interval.For the fourth immunization,100?g/one pure water mixed antigen was injected intraperitoneally.After 4 days,mouse spleen cells were collected and fused with SP2/0 cells.Indirect immunofluorescence assay(IFA)was performed on the supernatant of fused cells by using HeLa cells cultured in 96-well plate infected with NDV as the detection antigen.The positive hybrid tumor cells were cloned and purified for 3-4 times,and then frozen after expanded culture.Finally a monoclonal antibody of Newcastle disease virus M protein(Mab)was obtained.The specificity test of the Mab showed that it could recognize the M protein of different NDV strains.3.M protein nuclear localization study:This Mab was used to detect the time phase changes of M protein in cells after NDV infection.It was found that the M protein produced by virus infection was mainly distributed in the perinuclear cytoplasm and in the nucleus in a punctiform aggregation distribution.The intracellular distribution of M protein was compared with that of M plasmid with nuclear localization signal(NLS)sequence and M plasmid with knockout NLS sequence after transfection.It was found that the nucleation of M protein depends on its own NLS sequence.In this study,a monoclonal antibody with immunofluorescence was prepared.It was found that the M protein produced by virus infection was distributed around and in the nucleus in a punctiform distribution.And the nucleation of M protein depends on its own NLS sequence.This phenomenon provides a basis for further research on the nucleation of NDV M.