| Bovine tuberculosis mainly caused by Mycobacterium bovis (M. bovis) is a chronic, wasting zoonosis which has a great harm to cow industry. Meanwhile, M. bovis is one of the most important pathogens for human tuberculosis. Wild animals play a most important role as host of M. bovis. Currently, the common strategy to prevent this disease is the "test-and-slaugter" policy for international and domestic. Therefor, accurate and prompt diagnostic methods are significantly needed to prevent bTB and to ensure the health of animals and humanbeing. For bTB detection, tuberculin skin test (TST) is the only authorized method, but PPD based TST has many disadvantages including that some antigens are shared with other Mycobacterium species and BCG, the operation is a time-consuming and labor-consuming process. Therefore, to develop a more specific and sensitive diagnostic method for bTB is an urgent task for bTB control. The experience from developed countries which successfully controlled the bTB supports this view. Some other methods such as IFN-y in vitro release assay and antibody detection assay were also recommended by OIE. One of the key points of all the methods is to choose sensitive stimulated antigens or diagnositic antigens.M.bovis secrete many antigens during the process of infection, and the sensitivity of antibody detection or IFN-y in vitro release assay was low in which one single antigen was used. It has been confirmed that the "cocktail" antigen used in detection could boost the sensitivity of serologic diagnostic methods. However, the expression and purification of the members of "cocktail" antigens must be carried out individually, which takes much time and effort. This may lead to the problem in terms of the ratio of various antigens and it is hard to carry out the standardized product, which may affect the quality control of the diagnostic methods.In view of these problems, the genes Rv3872, CFP10 and ESAT6 in RD1 which was deleted in BCG were selected to produce fusion protein Rv3872-CFP10-ESAT6 (RCE) in the study for development of differentiated methods. At the same time, another fusion protein Rv3872-MPB70-MPB83-CFP10-ESAT6 (R5) was produced by introducing the Rv3872 into the previously fused four genes 70-83-CE in order to provide antigens with more sensitivity and more specificity. Two detection methods of RCE-indirect ELISA and RCEbased colloidal gold test strips were established and applied. The main results are summarized as follows: 1. Construction of the two expression vectors, the expression and purification of the antigensThe genes Rv3872, CFP10 and ESAT6 in RD1 were cloned, and two expression vectors pET-28a-RCE and pET-28a-Rv3872-70-83-CE were constructed. Protein RCE and R5 were expressed and purified with affinity chromatography. As a result, the purified RCE was obtained with the high yield. However, the purification of protein R5 was hampered probably because of high GC and gene redundancy It was hard to remove the un-related components in the protein.2. Character analysis of two fusion proteinsELISA and Western blot analysis showed that two fusion proteins both had specific reaction with positive antiserum against M.bovis, and had no reaction with positive sera of M. paratuberculosis, brucellosis, babesia and infectious bovine rhinotracheitis. Heat stability test established that two fusion protein had similar thermal stability, and the activity of them lossed quickly after treatment under 60℃for 1h. The primary application of two fusion protein in ELISA showed that RCE had a higher sensitivity than CE, but R5 had a sensitivity far lower than 70-83-CE because of some antigen sites overlapping with each other.3. Establishment of RCE-indirect ELISA and preparation of colloidal gold test stripsChessboard matrix titration showed the optimized conditions of RCE-ELISA was as follows:the optimized coating concentration of RCE was 15.625 ng/mL, blocking solution was 5% non-fat milk powder. The serum was 1:100 (volume ratio) diluted and HRP-labelled second antibody was 1:10000 diluted; 0.25% HF teminated the reaction after of the substrate DAB and HRP for 10 min.The optimal labeling pH of colloidal gold solution was 8.0. The optimal amount of RCE for colloidal gold labelling was 224μg per milliliter. The optimal concentrations of coating anti-RCE IgG for quality control line was 3.0mg/mL, and the coating protein RCE was 1.4mg/mL. The strips did not react with positive serum of other unrelated bovine diseases displaying a high specificity. Comparison test of the same positive antiserum to M. bovis showed that the sensitivity of the home-made strips was 4 times higher than that of commercial MPB70 test strips from Anigen, Korea in detection of a same positive serum of M.bovis. 4. Application of RCE-indirect ELISA and RCE-colloidal gold test strips in detection of clinical bovine tuberculosis and cervus tuberculosis323 clinical bovine sera were detected with the RCE-indirect ELISA, the total agreement with TST was 79.57%. RCE-indirect ELISA detected 22 positive samples more than CE-indirect ELISA, with a higher positive coincidence of 18.49%, and 26 nagtive samples more than CE-indirect ELISA, with a higher negative coincidence of 12.74%.301 clinical bovine sera were detected by RCE-colloidal gold test strips in parallel with commercial strips from Anigen, Korea. As a result, it had a positive coincidence of 97.78%(88/89), negative coincidence of 95.91%(211/220), total coincidence of 96.45% (299/310). It suggested that RCE-colloidal gold test strips had similar sensitivity and specificity to the commercial strips from Korea.468 Cervus Nippon sera were detected with RCE-indirect ELISA,6 more samples were detected positive and had total agreement of 99.35% to CE-indirect ELISA.100 clinical samples were detected by RCE-colloidal gold test strips,5 from 9 positive sera were detected with positive ratio of 5%, it showed that the sensitivity of RCE-colloidal gold test strips was lower than RCE-indirect ELISA (9%).In summary, RCE based ELISA and colloidal gold test strips improved the sensitivity in detection of clinical samples compared to CE based methods. Thus these modified methods would have better perspective in the program of bTB eradication.5. Application of RCE-indirect ELISA and RCE-colloidal gold test strips in detection of human tuberculosisBoth methods detected 25 sera from 32 sera from people determined to be tuberculosis patients with a positive ratio of 78.13%(25/32). In addition,18 sera from 22 sera were detected for the people determined by persistent infection by IFN-y in vitro release assay with a positive ratio of 81.82%(18/22). The results showed that RCE-indirect ELISA had good promise in detection of human Mtb infection. |
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