Expression Of Recombinant Fusion Proteins Of Mycobacterium Bovis And Their Application In The IFN-γ Release Assay

Bovine tuberculosis, caused by infection with Mycobacterium bovis, affects both human beings and domestic animals, which has been recognized as B infection by OIE. The prevalence of the disease not only influences the development of animal husbandry, but also threatens the health of human beings. However, the traditional methods, tuberculin test, can't diagnosis bovine tuberculosis rapidly and effectually because of its subjectivity, time-consuming, heavy work load, low specificity and senstivity. The interferon gamma assay is the most potential diagnostic method. But the use of bovine PPD as its stimulus induces high false-positive rate and low specificity. So the development of new specitific antigens to improve the interferon gamma assay is very imperative.This study focuses on the cloning of four antigen genes of M.bovis, the construction of fusion recombinant plasmids and the expressing of fusion recombinant proteins as the stimulant of the interferon gamma assay to enhance both the specificity and the sestivity. Four protective antigen genes of M.bovis, ESAT-6, MPB63, HSP65 and HSP70, were amplified from M.bovis genome DNA by PCR. The 288bp, 480bp, 1623bp and 1878bp gene segments were obtained. The PCR products were cloned into pEASY-T1 cloning vector. The identification by enzyme digestion and sequence analysis showed that the recombinant plasmids were constructed successfully and that these four genes are all the same as published data. The ESAT-6, MPB63, HSP65 and HSP70 genes were directed cloned into pET-32a(+) vector to construct recombinant fusion plasmids pET-E6-M63-H65 and pET-E6-M63-H70. Sequential analysis showed that the recombinant plasmids contain the complete ORF of ESAT-6,MPB63 and HSP65 or ESAT-6,MPB63 and HSP70. The recombinant plasmids were transformed into E.coli BL21(DE3), and then induced by IPTG. The SDS-PAGE analysis indicated that these fused target proteins such as 108kD and 116kD were acquired respectively. The target proteins were isolated and purificated by HisLink? Protein Purification Resin. After purification the two target proteins showed a specific band respectively on the SDS-PAGE. The results of Western-blotting analysis indicated that the two proteins were all antigenic.The IFN-γexpression level was detected after the whole blood of test cow were incubated with the two target proteins, ESAT-6-MPB63-HSP65 and ESAT-6-MPB63-HSP 70 to judge those tested cow are healthy or not. The results showed that the new methods had a high specificity of 100% and could discriminate one is infected with M.bovis or infected with M.avian, BCG vaccined or healthy. The new methods not only decrease the work load and the subjectivity of the judgement of the results, but also avoid intruding experiments. The new methods could be executed in 24 hours and repeated within a short time. Subject to the experimental condition, the data for sensitivity analysising is lack.