Immunological Evaluation Of Mycobacterium Tuberculosis Subunit Vaccine

Tuberculosis(TB) is an ancient disease which exist in17th century.During recent years, because of the huge number of infected people, increasing case of anti-drug TB and serious HIV/TB co-infection, TB becoming a big threatto the world. For take precautions against TB, BCG have been used80years ago but it is not an ideal vaccine yet. With the development of Biotechnology, a lot of new vaccines have been develop, including recombinant BCG, attenuated live vaccine, peptides and DNA vaccine. They have different advantages, but it still need clinical test further. It is the most effective method to prevent infection of TB with using vaccine to immunology public and control the locality immergence. TB vaccines mainly conclude two kinds:live attenuated mycobacteria and subunit vaccine. Although subunit vaccine doesn’t have more immunogenicity than live attenuated mycobacteria, it’s not effected by previous environmental mycobacteria and M.TB, most of all, it’s safe and simple to use for strength the adult immunology. After all, it’s an ideal vaccine. Subunit vaccine concludes protein subunit vaccine, DNA vaccine and recombinant virus subunit vaccine[1,2]. Protein subunit vaccine are advantage in terms of safety. It’s the most easy to be accepted by public. Nowadays, there have been more than20antigens have been found to induce the immunological protection against M.TB[3,4].Excretive antigen Ag85a belongs to Ag85complex which is considered have a good immunogenicity effect. M.TB is a kind of macrophage parasite bacteria which is closely related with the immune system. Cellular immunology plays the main role in the immunology against M.TB. In the infection M.TB, CD4+and CD8+T cell are the most important; the cytokines released from activated T cell, such as IFN-y and IL-2, which contribute to defeat against M.TB importantly.We cloned the Ag85a antigen from H37Rv genome and murine cytokines IFN-y and IL-2. We made the structure of recombinant plasmid of pet28a-Ag85a-IFN-γ和pet28a-Ag85a-IL-2, expressed and purify the two recombinant proteins in E.coli BL21cells. C57BL/6mice were immunized by Ag85a-IFN-γ and Ag85a-IL-2without signal peptide third time by subcutaneous at of2-week intervals. After two weeks of the last immunization, lymphocyte from spleens and the serum were detected for assessing the protection of the immunity for searching for the better vaccine for strengthen after prime-boost. The methods to evaluate the protective efficacy with detection of serum antibody(1gG,1gG1,1gG2c) by ELISA and percentages of lymphocyte subsets by flow cytometry. The results demonstrate that antibodies(1gG,1gG1,1gG2c) of fusion protein are more than antigen alone and the ratio of1gG2c/IgG1lead to TH1celluar immunity. CD8+/CD4+of flow cytometry means the fusion proteins have the better protective effect than Ag85a.We made the structure and expressed the recombinant fusion protein Ag85a-IFN-γ and Ag85a-IL-2, both of them have a better protection efficacy than Ag85a alone, and especially Ag85a-IL-2is better than Ag85a-IFN-γ...