Application Of Method To Detect Antibodies To M. Bovis In Cervus Nippon

Mycobacterium bovis results in bovine tuberculosis (bTB) in a range of animal species and humans. It is considered as B type infectious disease by world organization for animal health (Office International Des Epizooties, OIE) and considered as one of list B infectious diseases in our country. Investigation of tuberculosis pathogens showed that M. bovis is one of important pathogens of human tuberculosis. Therefore, the expert in World Health Organization (WHO) committee once said that tuberculosis can not be controlled unless bovine tuberculosis is eradicated.Due to the lack of an effective vaccine and medicine that could prevent and treat bovine tuberculosis, many countries adopt the "'test-and-slaugter" policy. So an accurate and timely diagnostic method is very important for preventing and controlling bTB. For the detection of bTB, tuberculin skin test (TST) is the only method recommended by OIE. Tuberculin is also called purified protein derivatives (PPD) which is a protein mixture that has complex components. PPD includes many antigens shared with other Mycobacterium species besides specific antigens of M. bovis. Hence, the PPD skin test can not avoid the cross reaction with other mycobacteria. Moreover the method propably provides false negative results when there is serious tuberculosis or immunosuppressive conditions. Therefore, to develop a specific and sensitive diagnostic method for bTB is an urgent task for bTB control.The farmed Cervus nippon with very high economy value is one of the special wild economic animals producing cornua cervi pantotrichum which is known as "one of three treasures in northeast China". However, Cervus nippon is considered as the secondary host of bTB. Recently, the epidemiological investigation about bTB in deer indicate that the risk coefficient of infection bTB is increasing in fanned deer. The infected deer with M. bovis will decrease the production of cornua cervi pantotrichum and emaciate which badly affect its economic value and the public health.In spite of the apparent disadvantage of TST, there are many restrictions for detecting bTB in wildlife such as deer with this test. The wildlife is difficult to capture and furthermore, there is no standard about the injection dosage and interpretation of the reaction to PPD. Therefore, to develop a specific and sensitive diagnostic antigen and diagnostic method for bTB is an urgent task for bTB control.M. bovis can induce the infected animals to produce antibodies to specific antigens. The serologic diagnostic methods by using single antigens is usually low in diagnosis of tuberculosis, but the combined antigens of M. bovis called "cocktail" antigens can dramatically increase the sensitivity of serologic diagnostic methods. However, for the "cocktail" antigens, the antigens have to be expressed and purified individually, which takes more time and effort. Moreover, this may lead to the problem in maintenance of the ratio of various antigens which may affect the sensitivity and specificity of the diagnostic methods. RV3872,CFP-10 and ESAT-6 are specific and secreted antigens of M. bovis and infected animals, but absent in BCG.Gold-Immunochromatographic assay (GICA) is a new immunological detecting technology since the early 1980s. It not only has good specificity and sensitivity, but also is cheap, simple and rapid.In the present paper, RV3872, CFP-10 and ESAT-6 genes were recombined, and the fusion protein RV3872-C10-E6 was efficiently expressed, in order to provide a sensitive and specific diagnostic antigen for bTB diagnosis. Besides GICA was developed by using fusion protein CE (cfp-10 and esat-6) to diagnose bTB. The main results were summarized as follows:1. Expression of fused cfp-10 and esat-6 genes of M. bovisThe fusion protein CFP-10 and ESAT-6 were expressed and purifed.. The properties of fusion protein were evaluated. ELISA showed that the protein could be recognized by the positive antiserum to M. bovis and mono-specific antisera to antigens CFP-10 and ESAT-6 respectively. Western blot analysis showed that the protein could be identified only by the antiserum to M. bovis but not the sera against M. paratuberculosis, brucellosis, babesia and infectious bovine rhinotracheitis virus. Heat treatment test demonstrated that the fusion protein was heat stable at 60℃for 1h . It was concluded that the fusion protein had antigenicity and was heat-stable. It would be a promising antigen used in M. bovis diagosis. 2. Establishment of colloidal-gold rapid detection testStrips were prepared with the principle of GICA with the fusion protein CE (cfp-10 and esat-6) . CE was labeled by colloidal gold and used as the test capture reagent to detect the corresponding antibodies. The highest sensitivity was found with strips prepared by 40 nm particles of colloidal gold. The optimal labeling pH of colloidal gold solution was 7.5. The best amount of CE for colloidal gold labelling was 0.96μg per ml. The optimal concentrations of capture reagent CE and control coating protein anti-CFP-10-ESAT-6 IgG was 3.0mg/mL and 2.5mg/mL (the antibody titer is 1:2¹⁰ ), respectively. The strips did not react with positive serum of other unrelated bovine diseases displaying a high specificity.3. Establishment of indirect ELISA using specific antigens of M. Bovis to Cervus nipponThe MPB83-ELISA, CFP10-ESAT6-ELISA and MPB70-83-CE-ELISA were developed by coating antigens MPB83, CFP10-ESAT6, MPB70-83-CE at the concentrations of 100ng/mL, 500ng/mL, and 500ng/mL respectively. Serum samples were diluted at 1:800, 1:200 and 1:200. The ELISAs based on these specific antigens had excellent repeatability.4. Clinical application of indirect ELISA and colloidal-gold rapid detection strips for specific antibodys of M. BovisA batch of colloidal gold test strips was manufactured to detect 637 clinical samples of Cervus nippon, in parallel with ELISA. The coincidence was high. This novel colloidal gold test strip for M. Bovis antibody detection is sensitive, specific, convenient to be used, rapid to get results. It would possess a good prospect in M. Bovis detection.5. The expression of fusion protein of RV3872, CFP-10 and ESAT-6Recovery the DNA fragments of RV3872 and pET28aCFP-10-ESAT-6 by Restriction enzyme digestion,then ligate the DNA fragments of RV3872,pET28aCFP-10-ESAT-6 and annealed double strands adapter by ligation system. After transformed DH5a the resultant recombinant plasmid was designated as pET RV3872-CFP-10-ESAT-6. The fusion protein was expressed as the soluble form by pET70-83-C10-E6 transformed BL21 (DE3) after induced by IPTG...