Expression of MAP0862-1595 Recombinant Protein of Mycobacterium avium subsp.paratuberculosis and Establishment of Indirect ELISA Detection Method
In order to establish a diagnostic method for the goat paratuberculosis based on the recombinant protein MAP0862-1595,this study screened two genes,i.e.,the MAP0862 and MAP1595,from the Mycobacterium avium subsp.paratuberculosis.The fu-sion gene MAP0862-1595 was obtained by using the overlapping extension PCR technology,subsequently linked to the recombinant plasmid vector pEASY-T1 and the recombinant plasmid vector pET30a,respectively.The reactogenicity of the recombinant protein of MAP0862-1595 was verified by using the Western-blot method,and the purified recombinant protein MAP0862-1595 was used as a coating antigen to construct an indirect ELISA method through optimization of reaction conditions.The results showed that the over-lapping extension PCR successfully amplified the fusion gene fragment of MAP0862 and MAP1595,with a length of 1 601 bp.The cloned recombinant plasmid pEASY-MAP0862-1595 and the recombinant plasmid pET30a-MAP0862-1595 were successfully con-structed,and the size of the recombinant protein of MAP0862-1595 was about 70 kDa,primarily existing in the form of inclusion bodies.The Western-blot analysis confirmed that this protein specifically bound to the positive sera of Mycobacterium avium subsp.paratuberculosis.The purified MAP0862-1595 protein was coated at a concentration of 1 p.g/well,with the primary antibody diluted at 1:400 and blocked with 5％skim milk,while the secondary antibody was diluted at 1:50 000,successfully establishing the ELI-SA detection method.The ELISA method demonstrated good sensitivity,specificity,and reproducibility,providing technical sup-ports for the detection of paratuberculosis and the development of kits.
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