| Tuberculosis (TB), a bacterial disease due to Mycobacterium Tuberculosis (M. tb).Mycobacterium bovis, the agent of TB in cattle, has been prevalent since ancient times. Despite being an ancient disease, tuberculosis (TB) remains one of the most devastating causes of morbidity and mortality worldwide. The magnitude of the disease reservoir is immense with about 2 billion people or one-third of the world's population thought to be infected with M. tuberculosis. In recent years, control of TB has been exacerbated by the deadly intersection of the HIV and TB epidemics and the emergence of multiple drug-resistant tuberculosis.The failure of current TB immunization procedures to adequately protect against M. tuberculosis infections is largely responsible for the unsuccessful global control of this disease. Although the current TB vaccine, M. bovis BCG, a vaccine against TB was developed by Albert Calmette and Camille Gu′erin in 1921, using a live attenuated strain of M. bovis, has widely used for at least 60 years, its efficacy has been shown to be highly variable in well-controlled clinical trials. While BCG is moderately protective against disseminated TB in children, BCG is ineffective in protecting against the most prevalent form of the disease, adult pulmonary TB. The inadequacy of BCG immunization to control the TB epidemic has stimulated a worldwide effort to develop more effective TB vaccination strategies.These immunization strategies have included live attenuated strains, viral vectored vaccine constructs and subunit formulations. These new approaches have potential advantages relative to BCG immunization such as increased safety, improved stability, and decreased interference by exposure to environmental mycobacteria. However, developing a vaccination strategy based on genetic or subunit vaccines that induces strong and sustainable anti-tuberculosis cellular immunity is a significant scientific challenge. Although immunizations with single antigen preparations have usually been inadequate for controlling intracellular infections, recent studies have shown that levels of pathogen specific cellular immunity can be substantially enhanced by combining different vaccines in a heterologous prime/boost. These heterologous prime/boost vaccination strategies often improve the magnitude and quality of T cell responses and can broaden epitope coverage. The subunit approach holds a number of advantages, such as increased safety and stability as well as the demonstrated ability to boost prior BCG. In addition, as subunit vaccines appear not to be influenced by environmental mycobacteria this type of vaccine. However, progress in this field has been delayed by the lack of adjuvant that induces a strong cell-mediated immune (CMI) response. These recent progresses can be partly attributed to the significant advances achieved within the field of adjuvant research.In this paper, ESAT6 gene and Ag85B gene were obtained by the basic operation of molecular cloning. Two genes connected through the linker as Ag85B-EAST6 fusion gene. Constructed pET-20b-Ag85B-EAST6 prokaryotic expression vector, transformed into BL21, expression of the success of the target protein with selected strain. Using His tag affinity chromatography method, the target protein was purified and the purified protein purity of more than 90%. The protein was identified by Western blot experiment. Used glycerol recovery solution on protein refolding, refolding rate was up to 90%.Experiment selected BALB/c mice as experimental models for the initial immunization use of rBCG, and Ag85B-EAST6 fusion protein were combined three adjuvant DDA/MPL, AD11.sm, ADO-1 to strengthen the immunity. Experimental results were evaluated by antibody titers, Th1/Th2 cytokines, IFN-r ELISPOT, CTL and other experiments. In the adjuvant DDA / MPL combined antigen Ag85B-ESAT6, found that DDA / MPL combined antigen Ag85B-ESAT6 immunization antibody titers of 1:25600, higher than the protein alone antibody titers after immunization to 1:12800, indicating DDA / MPL to enhance the intensity of the humoral immune protein promoted. More importantly, the DDA/MPL combined antigen Ag85B-ESAT6 of immunization, with Ag85B protein, ESAT6 protein and 9-2 peptide, 18-2 peptide, have emerged in response to stimulation by IFN-γin the ELISPOT experiments. While use of the protein alone and adjuvant alone had no spots, indicating the protein combined adjuvant had a significant immune response after immunization. During the prime-boost test, antibody titer of rBCG was 1:1600, rBCG + Ag85B-ESAT6 + DDA / MPL group is 1:25600, and the same as Ag85B-ESAT6 + DDA / MPL group after immunization. Moreover, other experiment groups of antibody titers were higher than rBCG. The rBCG group of IFN-γcontent 2000pg/ml was 4 times higher than the PBS group. Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group of IFN-γlevels compared with the PBS group increased by 11 times, 19 times and 7 times. IL-2 levels in supernatants of spleen cells with lower levels overall. Compared with the PBS group, rBCG group, rBCG + Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm Group of IL-2 contents have increased in more than 2 times, 4 times and 5 times. ELISPOT results showed that, the number of spots of rBCG +Ag85B-ESAT6 + DDA / MPL group, rBCG + Ag85B-ESAT6 + AD11.sm group and rBCG + Ag85B-ESAT6 + ADO-1 group were 5.6 times, 6 times and 3 times higher than rBCG group. After 20 peptide stimulation, the three groups produced 97,100 and 10 spots respectively. After 9-1 peptide and 9-2 peptide stimulation, rBCG +Ag85B-ESAT6 + AD11.sm group produced 33 spots and 43 spots. After stimulation with the Ag85B protein, activated CD4 + cells of rBCG +Ag85B-ESAT6 + DDA / MPL group and rBCG + Ag85B-ESAT6 + AD11.sm group were doubled than stimulation before. CTL results showed that each group of target cell for specific killing rate of less than 10%, indicating that specific CD8 + induced killing is not prominent.In a word: priming with rBCG and boosting with Ag85B-ESAT6 fusion protein combine with adjuvant were better than rBCG used alone. The adjuvant enhanced the immune response caused by the CD4 + T cell response, the CD8 + T cell response was weak. AD11.sm combined protein triggered stronger CD8 + T cell response than DDA / MPL and ADO-1. DDA / MPL was a pronounced effect kind of adjuvant, the experimental studies have shown AD11.sm adjuvant slightly better than DDA / MPL. Ag85B-ESAT6 fusion protein combined adjuvant as vaccine was better than rBCG, not only lead to humoral immunity, but also caused cell immunity, but mainly in CD4 + T cell response and CD8 + T cell response was relatively weak. |
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