Study On The Indirect-Elisa Diagnosis Of Paratuberculosis In Cow And Alpaca

Paratuberculosis is a chronic infection disease of cow, sheep, camelids and other animals caused by Mycobacterium avium subspecies paratuberculosis(Map). It is widely distributed in the whole world and could cause enormous economic lost. OIE compartmentalized the disease a kind of infection disease B. Until now, there is no treatable measure for it. The main way to prevent and cure it is detection, segregation and elimination. So, it is necessary to establish a sensitive and rapid method to detect this disease. It is reported that IS900is the specific gene of Map. Based on IS900gene, our researchers have done a serial of studies on establishment and application of indirect-ELISA diagnosis of paratuberculosis in cow and apaca.1. Prokaryotic expression of Map IS900gene and antigenicity analysis of the expressed productIn this study, the genomic DNA of Map K-10was extracted. IS900gene was amplified by PCR, and cloned into pGEM-T vector. The target gene was inserted into prokaryotic expression vector pET-32a(+) after being confirmed by PCR and restriction endonuclease analysis. The recombinant plasmid was transformed into E.coli BL21(DE3). The result of SDS-PAGE analysis indicated that the recombinant proteins could be expressed in inclusion body form at a high level in the presence of IPTG. The western blot analysis and ELISA showed the good antigenicity of the recombinant protein.2. Establishment and application of indirect-ELISA diagnosis of paratuberculosis in cowThe indirect-ELISA for detection of antibodies of Map in cow was established using recombinant fusion protein pET-32a-IS900as coated antigen. The result showed that the optimal concentration of coated antigen was4.4μg/mL, the best sealing condition was under24h with2%gelatin at4℃, optimal dilution of serum was1:100, optimal dilution of second antibody was1:10000, and the best substrate reaction condition was at37℃for15min. The positive criterion of the ELISA assay was OD490nm≥0.52. The cow serum stored in our lab were tested by the indirect-ELISA. We established the ELISA can be used for the detection of Map in cow as a rapid screening tool.3. Establishment and application of indirect-ELISA diagnosis of paratuberculosis in alpacaThe indirect-ELISA for detection of antibodies of Map in apaca was established using recombinant fusion protein as coated antigen. The result showed that the optimal concentration of coated antigen was3.75μg/mL, the best sealing condition was under24h with2%gelatin at4℃, optimal dilution of serum was1:50, optimal dilution of SPA was1:5000, and the best substrate reaction condition was at37℃for15min. The positive criterion of the ELISA assay was OD490nm≥0.62. The apaca serum stored in our lab were tested by the indirect ELISA. We established the ELISA was specific and sensitive, and can be used for routine diagnostic, which showed its better application prospection.