Preparation Of The Monoclonal Antibody Against EF-Tu Protein And Identification Of The B-cell Epitope In The Brucella

Brucellosis is a bacterial disease characterized by long-term fever,hyperhidrosis,arthritis,lymph nodes and hepatomegaly,and it also easily causes the disease of animals,such as abortion,infertility and orchitis,caused by the intracellular bacteria of the genus Brucella,and is still a serious agriculture,health and economic problems in many parts of the world.In recent years,the epidemic trend of brucellosis is becoming more and more serious.Our country is not optimistic about the form of prevention and control of brucellosis.Bacterial EF-Tu participates in the synthesis of protein and mediates the entry of amyl-tRNA into the ribosome space.It is an important extension factor that exists widely in the prokaryotes and is highly conserved.Recent studies have shown that EF-Tu is an important multifunctional protein,not only as a protein translation factor,but also,more importantly,that EF-Tu participates in many important cell and disease processes,including signal transduction,translation control,apoptosis,cytoskeleton composition,and so on.But few studies have shown the role of this protein in Brucella.An important reason is that there is no special tool to identify this protein.In order to further explore the role of EF-Tu protein in the bacterial infection process,we successfully expressed EF-Tu protein and prepared the monoclonal antibody of Brucella melitensis EF-Tu.We immunized BALB/c mice with the EF-Tu protein expressed by the prokaryotic expression.After the antibody titer of the immune BALB/c mice reached the standard of monoclonal antibody preparation,1 positive hybridoma cells that secreted monoclonal antibodies were obtaind by cell fusion,indirect ELISA screening and 5 sub clones,named BD6.Moreover,the epitope ¹¹⁰QTREHIL¹1616 of the epitope was screened and identified by peptide scanning.Bioinformatics analysis showed that the antigen determinant region was located in a highly hydrophilic region and formed part of Alfa’s helicoid.Finally,we used BD6 MAb and its antigen epitopes to detect Dot blot in 50 cases of clinical bovine serum.Compared with RBPT and ELISA,the positive coincidence rate of Dot blot detection results was 100%,the negative coincidence rate was 95%.These findings will provide useful information for the study of the antigen structure of brucellus,and may be of great value for the development of the antigen determinant vaccine of brucellus infection or for the diagnosis of brucellus.It provides a powerful tool for the study of the potential function of brucellus EF-Tu protein.