Screening Of New Molecular Markers For Tuberculosis And Vaccine

Tuberculosis (TB) is a chronic wasting zoonotic dis ease is mainly caused by Mycobacterium tuberculosis (M.tb) and Mycobacterium bovis (M.bovis). It not only makes huge economic losses to animal husbandry, especially dairy industy, but also is a great threaten to human health. Besides, TB can be demonstrated to spread between human and animals, and most of them were turned to be persistent infection. As the unique vaccine that is offically allowed to be used worldwide, BCG can only be protective on children but not adults. The diagnostic methods for tuberculosis currently include clinical chest X-ray and bacterium stain and isolation. but the chest X-ray test is a non-specific method. Because Mtb is slow-growing bacteria, the bacterial culture is a time consuming method and the detection rate for bacterial isolation is usually low. Besides bacterial culture and stain need very high level of laboratory biosafey. Molecular methods always need very special equipment and the rate of false positivity is high. Thus immunological methods are paid a great attention. The immune response in TB is characterized as the seperation of cellular and humoral immunity. At the early stage, cellular immunity is dominant, as the disease developed, humoral immunity will surpass. The conventional PPD skin test displays the cellular immunity. Although it has been used over a hundred years, it can not differentiate the infection between Mtb and mycobacteria of non-Mtb and BCG. IFN-γin vitro released assay was used abroad, but at primary stage, the antigen that was chosen for the stimulation was PPD, which has disadvantages as previously mentioned. Although the antigen was later changed to specific protein CFP10/ESAT6, the procedure is complicate because it needs to seperate lymphocytes from the whole blood. Furthermore, the price is very high, can not be accepted by common people in China. In this study, the IFN-y in vitro released assay was developed based on specific Mtb protein and simplified procedure. In order to discover novel molecules which might be potentially used in the diagnosis and vaccine of TB, we screened the specific proteins expressed in active tuberculosis by using cytokine microarray and protein microarray chips. By using the recognized molecular markers which have good antigenicity, a modified BCG was constructed and the immune response in mice was detected. The main results were summarized as follows: 1. Sceening of new molecular markers of TBSpecific protein CFP10/ESAT6 was used to stimulate the whole peripheral blood and the RayBio cytokine microarray chips were used to scan 516 signals including 510 cytokines in the supernatant. There were 67 cytokines up-regulated more than 1.9 folds and 43 down-regulated 2.0 folds after CE-stimulation. Including inflammatory factor, immume regulatory factor, chemotatic factor, growth factor, soluble receptor, adherence factor.In addition, SpringBio microarray was used to scan 656 signals including 651 proteins in the plasma of active TB patients, persistent infection persons, lung cancer patients and health control. In SpringBio array, only 21 proteins were increased and 72 decreased only in active TB patients.Proteins increased including keratin, neurofilament protein, proteins associated with gene repair and heat shock protein.2. Cloning and expression of human IFN-γAccording to the sequence published in GenBank (No. NM000619), the gene of intact squence of IFN-γwas synthesized, cloned into pET28a vector to construct the recombiant expression plasmid of hIFN-γ-pET28a. The plasmid was transferred into BL21(DE3) and highly expressed. The product was demonstrated to exsit in inclusion body.3. Development of hIFN-γmonoclonal and polyclonal antibodiesBy using recombiant protein hIFN-γ-pET28a as the immune antigen, the monoclonal antibodies and rabbit hyper-immune serum against hIFN-γwere developed. The monoclonal antibody titer was more than 215, while polyclonal antibody reached 212. Subunit identificatio showed the monoclonal antibody was IgG1.4. Establishment and application of IFN-γin vitro release assayA sandwich ELISA method to detect IFN-γwas developed by using the monoclonal antibodies as the coating antibody and the polyclonal antibody as the detection antibody. The CFP10/ESAT6 was used to stimulate the whole blood cells overnight and the supernatant of the culture was taken and detected for IFN-γ. This method was used to detect the TB patients and clinically health people. The results showed that the sensitivity was 94.6%(105/111). In 284clinically health people,236 were detected negative.5. Construction of the recombinant BCGThe genes Ag85B, Rv3872, CFP10 and ESAT6 were fused, cloned into the integrated vector pTIC6a, transferred into BCG and Mycobacterium smegmatis (M.S.) respectively by using electrotransformation method. The recombinant bacteria were immunized in mice. The serum antibody of mice plasma and peripheral blood lymocytes poliferation were tested. After 10 weeks post-innoculation, the IFN-γand IL-4 levels secreted by spleen cells in vitro were detected. As a result, after 4 weeks, rBCG group produced higher levels of antibody than BCG group (P=0.022). The spleen cells proliferated more effective than the BCG group. After 10 weeks of immunization, the rBCG group yielded significantly higher levels of IFN-y than rBCG. These findins showed that rBCG stimulated both significantly higher cellular and humoral immune response. Although rM.S. could induce immune response either, the extent was weaker and duration was shorter than that in rBCG and BCG groups.