Preliminary Screening Of BHK-21 Cells Receptor Protein In 43 KDa OMP Of Bovine Fusobacterium Necrophorum

Fusobacterium necrophorum is a gram-negative non-spore specific anaerobic pleomorphic bacteria,which is a common bacteria in human and animal digestive tracts.One of the most important factors in the pathogenesis of Fusobacterium necrophorum is the adhesion of bacteria to host cells.The adhesion of the bacteria to the host cells helps it to colonize the host tissue cells,then propagate within the cells and spread to other tissues.Although studies have shown that the43 k Da outer membrane protein(OMP)of Fusobacterium necrophorum can adhere to hamster kidney cells,bovine endometrial cells and bovine mammary epithelial cells,it is unclear which protein or which series of proteins in the host cell can interact with it.Therefore,this experiment screened the interacting proteins of the 43 k Da OMP and BHK-21 cells of Fusobacterium necrophorum.In this experiment,the eukaryotic expression vector of p CAGGS-43 k Da OMP was constructed,and then transformed into E.coli for plasmid extraction.The extracted recombinant plasmid was transfected into BHK-21 cells for transient expression,the monoclonal antibody of43 k Da OMP of Fusobacterium necrophorum was prepared,and the titer of monoclonal antibody was determined by indirect ELISA.Subtype identification,Western blot identification and indirect immunofluorescence identification were performed.Finally,the monoclonal antibody of the prepared 43 k Da OMP was used to screen the protein interacting with BHK-21 cells by the43 k Da OMP of Fusobacterium necrophorum by co-immunoprecipitation.The results are as follows:(1)The 43 k Da OMP of Fusobacterium necrophorum was transient expressed in BHK-21 cells.(2)A monoclonal antibody against 43 k Da OMP of Fusobacterium necrophorum was successfully prepared and named O43.(3)Differential bands were screened by co-immunoprecipitation method,and 70 suspected differential proteins were detected by mass spectrum.This study initially screened the protein interacting with BHK-21 cells of the 43 kDa OMP of Fusobacterium necrophorum,and provided a premise for the study of the protein interacting with the bovine host cell of the 43 k Da OMP of Fusobacterium necrophorum,and further understanding of Fusobacterium necrophorum.The pathogenesis provides more possibilities and provides a new theoretical basis for the design of antibacterial drugs for Fusobacterium necrophorum.