Expression Of Fragments Of MO P60 And P65 Proteins And Establishment Of A Recombinant Protein P60(n)-P65(C)Based Indirect ELISA

Mycoplasma ovipneumoniae has played a major role in the respiratory diseases of sheep and goats.Screening the key immunodominant proteins and establishing an indirect ELISA with high sensitivity and specificity are significant for clinical diagnosis.In this study,the truncated membrane proteins p60 and p65 of MO were expressed using the prokaryotic expression system.The N-terminal part and C-terminal part of p60 and p65 genes were cloned by PCR separately;Both p60(C)and p65(N)have a TGA condon.So the overlap-extension PCR was used to mutagenate nucleotides TGA to TGG;Then the PCR products were inserted to the pET-32a(1)vectors for expression in E.coli BL21(DE3);Western blotting was conducted for analysis of expressed products;Four purified recombinant proteins and the whole-cell proteins were used to coat onto the microtiter plate wells,then thirty sheep serum samples were tested.Western blotting revealed that the expressed recombinant p60-N,p60-C,p65-N and p65-C could specifically react with positive serum.Comparing with the results of iELISA using the whole-cell proteins as antigens,the recombinant proteins p60-N and p65-C were conformed to be the optimal immunogenic proteins.On the basis of these results,the p60-N and p65-C genes were spliced with overlap extension RCR.Then the tandem recombinant was expressed in E.coli.Western blotting was conducted for analysis of expressed products.Then an indirect ELISA was established using the tandem recombinant p60(N)-p65(C)protein as the coating antigen.And the reaction conditions have been optimized.Then 150 serum samples were tested using this iELISA system;Moreover,this iELISA was used to detecting the antibody titers of serum of which have been immuned by inactived MO vaccines;Then comparing with the results of iELISA using the whole-cell proteins as antigens.The results of testing serum samples using these two methods showed that a coincidence rate of 90.67%,and the two methods were carried out by testing 40 murine serum samples,which showed a good consistency of the serum antibody titers.Taken together,the study using the tandem recombinant p60(N)-p65(C)protein as coating antigen for the indirect ELISA for detection of antibody against MO.The study provided a basis in developing the commercial ELISA kit to detect the disease.