Expresion Of The P28 Gene Of Mycoplasma Bovis And Development Of The Indirect-ELISA

Mycoplasma bovis is an important pathogen of pneumonia, arthritis and so on in calves. It was generally estimated that M.bovis contributed a lot to the economic lose in cattle industry. The first report about pneumonia caused by M.bovis in Chnia was recorded in the year of 2008. The pathogen has been an increasingly important factor that threatens the cattle industry in our country. However, the absence of an international reference test to diagnose M.bovis poses difficulty to the disease control. This study established the M.bovis antibody enzyme-linked immunosorbent assay (ELISA) and developed the ELISA kit to fill the emptiness of M.bovis antibody ELISA kits inside.First-phase preparations for the study include some aspects as follows. First, the sequencing of the whole genome of M.bovis Hubei isolation was finished. Second, we obtained 58 presumed membrane protein genes after analyzing of the whole genome via bioinformatics soft. Third, we obtained the recombinant proteins by applying prokaryotic expression system, and then screened the recombinant proteins by western blotting. Finally, we obtained an immune associated recombinant protein named P28.In the study, the recombinant protein P28 was obtained by applying prokaryotic expression system. M.bovis antibody ELISA kit named as MbH kit was developed based on the recombinant protein. Firstly, technical indicators of MbH kit such as sensitivity, specificity, accuracy, antibody duration, repeatability, stability were evaluated. The results showed that the sensitivity of the kit was 93.04% and the specificity of the kit was 97.80%. Morover, there was no cross reaction with positive sera of other diseases in cattle. The totle agreement of the kit to pathogenic diagnosis and serodiagnosis was 95.41%. The antibody of infected calves could be detected in 1 week after infection and the kit could be available in 20 weeks after infection. CVs of intra-plate and inter-plate were lower than 10%. The kit storaged in 4℃was stable in 14 months. Secondly, MbH kit was compared to commercial kits, kit 1 and kit 2, on the indicators of sensitivity, specificity, accuracy and consistency. The results indicated that the sensitivity of MbH kit was as good as the commercial kit 1. The specificity test results indicated that MbH kit and kit 1 have no cross-reaction with international standard serum of contagious bovine pleuropneumonia, but it was positive by commercial kit 2 detection. The detection agreement determinate by comprehensive diagnosis of results of the MbH Kit and the Kit 1 were 92% and 96% respectively, while the agreement of the Kit 2 was merely 74.67%. The Kappa consistency check of the three kits indicated that there was strong consistency between MbH kit and Kit 1 while there was moderate consistency between Kit 2 and MbH kit or between Kit 2 and Kit 1. In conclusion, MbH Kit and Kit 1 were recommended as reliable method both in routine diagnostics and in epidemiological survey.