Fusion Expression And Immunogenicity Of Mycoplasma Ovipneumoniae P60 Gene And Mouse IgG Fc Gene

Mycoplasma ovipneumoniae(Mo)is one of the most important opportunistic pathogens causing interstitial pneumonia in sheep.Although the mortality caused by Mo is not high,the epidemic scope is wide and the onset period is long,especially for lambs.In this study,an indirect ELISA method was established to detect Mo antibody level through prokaryotic expression of the main protective antigen P60 protein,and P60 gene was fused with mouse IgG Fc fragment.Fusion proteins were obtained by prokaryotic expression and the immune protective effect of the protein was studied.The main results are as follows:1.The main protective antigen P60 gene and mouse IgG Fc gene of Mo NM2010 strain were amplified respectively,and the P60 gene was fused with mouse IgG Fc gene by SOE-PCR method.Finally,P60 protein and P60-Fc fusion protein were obtained by prokaryotic expression.2.Using P60 protein as diagnostic antigen,an indirect ELISA method was developed for the detection of serum antibodies against Mycoplasma ovipneumoniae.The concentration of coated antigen was 0.5μg/m L,the optimal dilution concentration of primary antibody was 1∶100,and the incubation conditions were 37 ℃ and 1.5 h.The total coincidence rate between the method and indirect hemagglutination method was88.0%.Therefore,the indirect ELISA method established is reliable and can be used to detect clinical samples,providing a reliable method for the diagnosis and epidemiological investigation of Mycoplasma ovipneumoniae.3.Using P60 and P60-Fc proteins as immunogens,the immune efficacy of P60 and P60-Fc proteins under different inoculation methods was evaluated by measuring the content of specific IgG in serum and local s Ig A in respiratory mucosa of mice,as well as observing the amount of bacteria in lung and pathological section of mice after challenge.The results showed that adjuvant combined with P60-Fc protein had the best immune effect by intramuscular injection and nasal drop respectively for the first and second immunization,which could not only induce the body to produce a good systemic immune response,but also cross the respiratory mucosal layer into the body and form an effective mucosal immune response locally,so that the mice could resist the invasion of Mo.