Abstract: 【Objective】 The purpose of this study was to establish an indirect ELISA method based on lipoprotein P60 of Mycoplasma ovipneumoniae.【Method】 Bioinformatics softwares were used to analyze the antigenicity of P60 encoded proteins,and the region with the best antigenicity was screened out.After the target gene was amplified,the recombinant expression vector was constructed and the gene was sequenced,the correctly sequenced plasmid was transformed into Escherichia coli BL21(DE3) competent cells.The expression of the best antigenity protein was induced by IPTG and the induction time was optimized.SDS-PAGE was used to analyze the molecular weight of the protein and its expression form.Using recombinant protein as antigen and positive serum of Mycoplasma ovipneumoniae as antibody,Western blotting was used to analyze its reactivity.An indirect ELISA method was established to detect the best antigenicity protein by using the recombinant protein as the coating antigen.Twenty negative serum samples were detected by this method,and the critical value of positive and negative was calculated.The specificity,sensitivity and repeatability of this method were tested.The established indirect ELISA method and indirect hemagglutination were used to detect 92 sheep clinical serum samples.【Result】 According to DNAStar analysis,the average antigenic index of 58-928 amino acid region of P60 protein ranged from 0.8 to 1.7,and hydrophilic index ranged from 0 to 2.0,indicating that the region P60_58aa-928aa had strong antigenicity and hydrophilicity.The P60_58aa-928aa gene was amplified by PCR,and the recombinant expression vector was constructed.Gene sequencing proved that the recombinant expression vector was consistent with the expected results.SDS-PAGE results showed that when IPTG was induced at the concentration of 1 mmol/L and the induction time was 10 h,the protein expression level was higher,the molecular weight of protein was 42 ku,and it was expressed as inclusion body in Escherichia coli.Western blotting results confirmed that the recombinant protein had good reactivity.The optimal ELISA conditions showed that the concentration of antigen coating was 0.5 μg/mL,the optimal dilution concentration of primary antibody was 1∶100,the optimal incubation time was 1.5 h,and the optimal dilution ratio of enzyme-conjugate secondary antibody was 1∶4 000.The critical value of positive and negative serum was 0.36.The standard positive serum of Foot-and-mouth disease virus (FMDV),Peste des petits ruminants virus (PPRV),Brucella,Mycoplasma bovis (Mb) and Mycoplasma filamentum goat subspecies (Mmc) were all negative,which proved that the method had high specificity.In the sensitivity test,the result was still positive at the serum dilution ratio of 1∶2 048.The coefficient of variation of intra-batch and inter-batch repeated tests were all less than 10%,which proved that the method had good repeatability.The established indirect ELISA method and indirect hemagglutination method were used to detect 92 sheep serum samples,and the results showed that the positive coincidence rate was 81.6%,the negative coincidence rate was 92.6%,and the total coincidence rate was 88.0%.【Conclusion】 This indirect ELISA method could be used to detect clinical samples,and providing a reliable method for the evaluation of vaccine immune effect and disease diagnosis of Mycoplasma ovipneumoniae.

Key words: Mycoplasma ovipneumoniae; P60_58aa-928aa protein; prokaryotic expression; indirect ELISA