Establishment Of Gold Immunochromatographic Method For Bovine Paratuberculosis

Bovine paratuberculosis is caused by M.paratuberculosis (MP) and it is a kind of chronic wasting disease in ruminants globally.This disease causes chronic and hyperplastic enteritis in ruminant -s,e.g.cattle,caprine,deer and so on,the cattle can be infected easily.The prevalence of bovine paratuberculosis showing a rising trend In China, it also caused tremendous economic loss.how to effective control bovine paratuberculosis has become a major issue in the cattle industry. Because it is difficult for the diagnosis of bovine paratuberculosis and conventional detection technology is not good ,the test results are not accurate.So it is necessary to establish a method (GICA) for the diagnosis of bovine paratuberculosis, this method was rapid and convenient and can be used widely.The three major antigens of M.paratuberculosis,MAP0862,MAP2154c were analysised by DNAstar.The epitope which is in a high antigenic index was Synthesised and obtained the fusion gene PGEM-map0862-2154c.Then the fusion gene was inserted into the pET28a(+) to construct the recombinant plasmid pET0862-2154c.The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express the fusion protein which was induced with 0.4mmol/L IPTG..The expressed fusion protein was in soluble in SDS-PAGE analysis and Weatern-blot detection.These resules demonstrated that the purified protein was 23.5 kDa(include 3.5 kDa pET28a(+)in molecular weight).The purified bovine recombinant protein of Mycobacterium paratuberculosis was immobilized on a nitrocellulose membrane detected line as antigen,bovine IgG was immobilized on a nitrocellulose membrane control line as antibody,colloidal gold labeled SPA was sprayed on glass cellulose membrane to prepare the colloidal gold immunochromatograpic strip. There was a red reaction strip at the test line only when detecting the standard positive serum,and the red reaction strips all appeared at the control line whatever.The test strips detecting antibodies had a minimum concentration of antigen 400ug/mL,which did not react with negative serum of bovine paratuberculosis, positive serum of bovine tuberculosis,bovine brucellosis, aftosa,bovine infectious rhinotracheitis,and the results of different batches of test strips saved in room temperature and 4℃were the same in four months.These results revealed that the test strip has good sensitivity,specificity,and stability, the result was observed at about 10min, this new method is a simple way and used for clinical testing.