Preparation And Identification Of Monoclonal Antibody Against NADH Oxidase Of Mycoplasma Synoviae

Mycoplasma synoviae(MS)is the major pathogen causing respiratory disease in poultry.MS infection in chickens or turkeys can cause airsacculitis,joint effusio synovitis,and infected chickens suffer from limping,reduced egg production,and physical deterioration,which can be transmitted vertically to future generations,causing huge economic losses to the poultry industry worldwide.Usually,the infection of MS is regional and large-scale outbreaks are rare.In recent years,due to the relatively poor prevention and control of MS in China compared with Mycoplasma galliscepticum(MG),MS infection has become more and more serious.NADH oxidase(NOX)is an important oxidoreductase that maintains the growth of biological metabolism.Studies have shown that NOX not only acts as a catalytic enzyme,it is also a membrane surface adheson in some pathogenic bacteria,which can assist pathogens in adhering to host cells and even affect virulence.Previous studies in our laboratory have shown that the NOX of MS not only functions as a catalytic enzyme in the cytoplasm but also exists in a small amount on the membrane surface of MS.It is immunogenic and a possible adhesion-related protein.In this study,the recombinant strain E.coli BL21(p ET28a-MSNOX),which was constructed in our laboratory,was induced and expressed by IPTG.Sodium dodecyl sulfate(SDS)-Polyacrylamide gel electrophoresis(PAGE)analysis revealed that the recombinant MSNOX(r MSNOX)protein was expressed in the supernatant and precipitate of the recombinant E.coli.The r MSNOX protein in the supernatant was purified with His-Tag magnetic beads to obtain a relatively pure r MSNOX protein.The purified r MSNOX protein was mixed and emulsified in equal volume with Freund’s adjuvant to immunize BALB/c mice.The mouse serum was collected and the antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).The spleen cells of the mouse with the highest antibody titer were fused with murine myeloma cells.The fused hybridomas cells were screened by indirect ELISA and limiting dilution method until a hybridoma cell line capable of stably secreting monoclonal antibody against r MSNOX protein was selected.The specificity of the mononclonal antibody was detected by indirect ELISA and Western blot..The obtained hybridoma cells were passaged 10 times and repeatedly frozen and thawed to test the stability of the antibodies secreted by the hybridoma cells.In this study,a hybridoma cell line capable of stably secreting monoclonal antibody against r MSNOX protein was successfully prepared,named 2F4.The results of indirect ELISA,Western blot and immunofluorescence assay showed that the monoclonal antibody can only react with the MS,but not with other poultry pathogenic bacteria.The monoclonal antibody is identified as Ig G2 b subclass and κ chain by using the monoclonal antibody subtype analysis kit.The hybridoma cell line has the ability to secrete antibodies stably after continuous passage and repeated freezing and thawing,indicating it has good stability.The selected cell line 2F4 was analized to have 98 ± 5 chromosomes,which was consistent with the chromosomes number of a hybridoma cell.The selected hybridoma cells were injected intraperitoneally into 6-8 weeks old BALB/c mice to prepare ascites,and the antibody titer of ascites reached 1: 2048000 by indirect ELISA.In summary,we expressed and purified the r MSNOX protein,and successfully prepared a hybridoma cell line that can stably secrete the monoclonal antibody against r MSNOX protein.The preparation of the monoclonal antibody may help for establishment of the MS detection method and the further study of the role of MSNOX in the pathogenesis of MS.