Research Protein-protein Interaction Between S1 Glycoprotein And Membrane Protein Of Avian Infectious Bronchitis Virus By Yeast Two-Hybrid System And Co-immunoprecipitation

Avian infectious bronchitis virus (IBV), a member of the family Coronaviridae, order Nidovirales, is a highly infectious pathogen of domestic fowl. S protein and M protein have played an important role in viral invasion and adsorption. The S1 subunit is involved in the induction of neutralizing, serotype-specific and haemagglutination inhibiting antibodies, and protective immunity. The M protein, a polytopic protein, is the most abundant component of coronavirions. When synthesized in the absence of other viral components, M protein tends to accumulate in the Golgi complex as detergent insoluble, polymeric structures, presumably as part of it sretention mechanism. The M protein as the major inducing component has been proposed to play a role in interferon induction. But now, is there have some interaction between S1 glycoprotein and M protein has not been reported.In this study S1 gene and M gene of infectious bronchitis virus was amplified by RT-PCR method and studied their abilities to interact with each other. The S gene was subcloned into pGBKT7 and The M gene was subcloned into pGADT7. 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-Sl and PGADT7-Rec-M grew on SD/-Ade/-His/-Leu/-Trp plates, and detection of the activity assays of the report gene lacZ's expression product-liquid-gal actosidase showed positive results. Cotransformation yeasts AH109 can make the nitrocellulose filter turn blue, to illustrate the reporter genes LacZ is activated to expression. To our knowledge, it is initial manifested the interaction between S1 protein and M protein in yeast cells.To explore the interaction between S1 and M by co-immunoprecipitation, construct the Myc-tagged fusion protein (Myc-M) in expression vector pCMV-Myc-M, HA-tagged fusion protein (HA-S1) in expression vector pCMV-HA-S1 into HEK293 cells. The interaction between S1 and M was detected by co-immunoprecipitation. Double restriction enzyme digestion showed that pCMV-Myc-M and pCMV-HA-S1 were constructed correctly. When Myc-M was immunoprecipitated by anti-Myc antibody, Myc-M was identified by Western blotting with anti-Myc antibody from immunoprecipitated complex. To study S1 protein secondary structure, M protein secondary structure by bioinformatics analysising to infer that interaction domain reside in flexibility and hydrophilicity area.This study successfully constricted the spinner expression vector PGBKT7-S1, prey expression vector PGADT7-Rec-M, recombinant expression vector pCMV-Myc-M and recombinant expression vector pCMV-HA-S1. The experiments demonstrate that the S1 protein and M protein in Avian infectious bronchitis virus have interaction by the technology of Yeast Two-Hybrid System and co-immunoprecipitation successfully.