The Preliminary Research Of T Lymphocyte Proliferation Inhibition Caused By Newcastle Disease Virus Infected Dendritic Cells

Newcastle Disease(ND) is a acute contagious infectious diseases, causing significant economic losses wordwide. The causative agent is Newcastle disease virus(NDV). Unfortunately, neither traditional control strategies nor conventional vaccines have provided sustainable control of ND. A major obstacle in the development of a successful ND vaccine is that highly variable NDV induces immunosuppression.Dendritic cells(DC) is the most powerful antigen presenting cells as known, can present antigen by pinocytosis, phagocytosis and receptor-mediated. DCs as a bridge that crosses from the natural immune and acquired immune, be able to play a regulatory role to antigen. Some virus can escape immune surveillance and increse infection by infecting DCs.DC-SIGN is a C-type lectin receptors on the surface of the DCs, belong to the type Ⅱ transmembrane protein, participate in identifying and intake of antigen, DC-SIGN is necessary in DCs adhesion, migration, inflammation, activating the primary T cells and trigger the immune response.To verify the effect of DC-SIGN on T lymphocyte proliferation when infected DCs by NDV and the main pathway of NDV enter DCs, we carried out the study of the following three parts.The first part: DCs were infected with the NDV virulent strain Herts/33 and low virulent strain NDV-GFP, infection can stimulates the maturation of DCs and also changed DCs surface molecules and cytokine expression, acquired the function of immune regulation. DCs can efficiently activate T cells, start the initial immune response.The second part, Using Transwell test showed the role of DCs in CD4+ T lymphocyte proliferation, demonstrated NDV live virus inhibiting T cell proliferation needed to rely on touch between DCs and T cells.Flow cytometry and Western blot experiments confirmed the DC-SIGN antibody of NDV affected the inhibitory role on T cell proliferation, showed the DC-SIGN exerted inhibitory functions during NDV infecting DCs and T cells.The effect on cells macropinocytosis inhibitor Rottlerin showed NDV infection DCs mainly by the way of macropinocytosis.The third part, we built the DC-SIGN stable expression cell lines, inserted the DC-SIGN gene into pcDNA3.1 eukaryotic expression vector, and transfected into HeLa cells, by G418 pressurized screen and limited dilution method to obtain a stable DC – SIGN expressing cell line. By PCR and Western blot methods validation, the cell line can correctly and stably expressing DC-SIGN protein, repeat cryopreservation and resuscitation and continuous subculture cells can still stable expression DC-SIGN protein, these verified cell lines were successfully bulit and establised foundation for the subsequent study of NDV viral proteins interact with DC-SIGN.