Identification Of The Immunogenicity Of Mycoplasma Bovis Membrane Proteins DnaK,L-LDH And P48

Mycoplasma bovis(M.bovis)is one of the most important and frequently isolated mycoplasmas that cause disease in cattle.It can clinically cause bovine respiratory diseases and related diseases such as arthritis,mastitis and reproductive disorders,which is a serious threat to the healthy development of the cattle industry.Due to its special structure and biological properties,as well as the characteristics of its co-infections and persistent infection,the pathogen will persist in the cattle for a long time,causing repeated epidemics of the disease.However,the clinical diagnosis and prevention of M.bovis have been facing a great challenge.Currently,there is neither a specific medicine nor a safe and effective commercial vaccine.Therefore,the development of specific,rapid detection methods and safe and available vaccines are still an urgent requirement in the diagnosis,treatment,prevention and control of M.bovis disease.Because mycoplasma has no cell wall,its adhesion,invasion,and pathogenic mainly rely on the cell membrane,and the main antigenic component on the cell membrane is Lipid-associated membrane proteins(LAMPs).The study of immunogenicity of LAMPs is of great value for the diagnosis and prevention of M.bovis disease.However,due to a large number of proteins in LAMPs,the screening range of proteins with good immunogenicity is wide,which is a heavy workload.The current knowledge of M.bovis LAMPs is still limited.Therefore,in this study we extracted the LAMPs of the M.bovis TJ strain and successfully divided it into 5 groups by AKTA molecular sieves,the screening range was narrowed without destroying the natural conformation of these proteins.Then the immunoreactivity identification assay was performed to screen out the specificity group,which is assayed by mass spectrometry.The mass spectrometry results were analyzed by integrating the protein matching score ranking order and gene annotation,three membrane proteins(Dna K,L-LDH and P48 protein)were initially screened,and then prokaryotic expression and purification of the three proteins were performed respectively.The result of the western blot experiment showed that the immunogenicity of the three proteins is good,which is subsequently evaluated by ELISA analysis.The ELISA results showed that the sensitivity and specificity of the P48 protein are good,but Dnak and L-LDH had cross-reaction with the other common bovine respiratory disease positive sera.Moreover,the immunogenicity of the three membrane proteins was further analyzed through the mouse immunization experiment.The results showed that Dna K,L-LDH and P48 can induce high-level and long-lasting serum antibodies in the mouse body,including Ig G1 and Ig G2a antibodies.Among them,the antibodies induced by the Dna K and L-LDH protein immunization group are biased towards the Ig G1 subclass,while the P48 protein is biased towards the Ig G2a subclass.Compared with the control group,the three proteins can enhance CD4~+but reduce the response of CD8~+T lymphocyte subgroups.The immunization dose has no significant difference in the level of antibodies and subclasses produced by the three proteins after immunization alone,but the differentiation of lymphocyte subsets in the L-LDH protein immunization group is significantly different.Compared with a single protein antigen immunization,three-antigen mixture immunization can induce a higher level of Ig G antibodies against P48 and a reduction in the level of Ig G2a antibody subclasses,but it has no significant effect on the differentiation of lymphocyte subgroups.In summary,this study used a molecular sieve and proteomics techniques to initially screen out three proteins with immunogenic and conservative from M.bovis LAMPs(Dna K,L-LDH and P48),among which the L-LDH protein is the first reported in M.bovis.These three proteins induce the body's lymphocyte response belongs to the CD4~+helper T lymphocyte immune response.Dnak and L-LDH proteins have a Th2 type dominant response,which induced a strong humoral immunity,while the immune response induced by the P48 protein has a Th1 type dominant response with the preference of inducing cellular immunity.The results of this study provide more antigen information for the early diagnosis of M.bovis and the screening of potential antigen proteins for the vaccine development.