Protein-protein Interaction Between Membrane And Nucleocapsid Protein Of Avian Infectious Bronchitis Virus Studied By Yeast Two-hybrid System And Co-immunopreciptation Method

Infectious bronchitis virus(IBV) is a world-wide viral disease of poultry,which poses a major economic threat to the poultry industry.As a result of IBV replication study of the molecular mechanism has not been clear how to effectively control the IB has become a major problem.Based on the initial work,we have amplified M gene and N gene of infectious bronchitis virus by PCR method and studied their abilities to interact with each other.The M gene was subclonedinto pGADT7,and the N gene was subcloned into pGBKT7.After being verified with restriction endonuclease digestion and DNA sequencing,the recombinant vectors were transformed into yeast cell AH109 by lithium acetate method. The yeast cells co-transformed with pGBKT7-N and pGADT7-M grew on SD/-Ade/-His/-Leu/-Trp plates,and the liquidβ-galactosidase activity assays showed positive results.We found that interaction of the N and M proteins takes place in vivo. These data provide useful clues for elucidating the molecular mechanism of the function of N and M.we constructed these fusion expressing plasmids in mammalian cells including pCMV-HA-N and pCMV-Myc-M.After being verified with restriction endonuclease digestion,expressing plasmids were transfected into human embryo kidney 293(HEK293)cells.Explore the interaction between N and M protein by co-immunoprecipitation.Double restriction enzyme digestion show that pCMV-HA-N and pCMV-Myc-M were successfully constructed,providing materials for further study. Plasmids were cotransfected HEK293 cell.Using anti-HA polyclonal antibody precipitation,then detect the expression of M protein by anti-Myc monoclonal antibody. The result showed the interaction between N and M protein in mammalian cell.we employed yeast two-hybrid system and Co-IP to investigate possible interactions between IBV nucleocapsid(N) and the membrane(M) proteins,which provided a base for further study on the evaluation of critical AA sites.It is necessary for the elucidation of the molecular mechanism of IBV replication and rationalization of anti-IB therapeutic intervention.