A Study Of T-cell Function Exhaustion Induced By Persistent Mycobacterium Tuberculosis Infection And Construction Of Fusion Protein AEMD And LT70

Part ⅠA STUDY OF T-CELL FUNCTION EXHAUSTION INDUCED BY PERSISTENT MYCOBACTERIUM TUBERCULOSIS INFECTIONObjective:To explore whether the mechanism, that chronic severe TB patients lower immunity against Mycobacterium tuberculosis, is the long-term infection sustained activation of the immune response caused by T-cell dysfunction or not.Methods:We collected 35 cases tuberculosis patients from Lanzhou Pulmonary Hospital, admitted in January 2013-December 2014, including 12 sputum smear-positive cavitary TB patients. We collected six healthy volunteers from Lanzhou University and isolated lymphocytes from obtained blood samples of experimental group and the control group. Analyze the level of T lymphocyte secreting IFN-γ and IL-2 by ELISPOT and ELISA. Analyze the ability of T cell proliferation by cell proliferation assay. Detect the expression of immunosuppression related molecules Tim-3 on CD4+, CD8+ T cell by flow cytometry.Results:The results showed that for sputum smear-negative TB patients, the ability of T cells proliferation was significantly enhanced comparing with the healthy donors. For sputum smear-positive cavitary TB patients, the ability of T cells proliferation was significantly decreased comparing with the healthy donors and sputum smear-negative TB patients. For sputum smear-positive non-cavitary TB patients, the ability of T cells secreting IFN-γ markedly increased comparing with the healthy donors. For sputum smear-positive cavitary TB patients, the ability of T cells secreting IFN-y was significantly decreased comparing with the sputum smear-positive non-cavitary TB patients. For sputum smear-positive non-cavitary TB patients and sputum smear-positive cavitary TB patients, the expression of immunosuppression-related molecules Tim-3 significantly increased comparing with the healthy donors. However, the expression of immunosuppression-related molecules Tim-3 had no significant difference for TB patients. For TB patients, the ability of T cells secreting IL-2 was significantly reduced comparing with the healthy donors, while there was no significant difference for TB patients. When grouped according to age the patients, there was no difference in the groups.Conclusion:For sputum smear-positive cavitary TB patients with persistent Mycobacterium tuberculosis infection, the function of T cells was waning. The proliferation of T cells was significantly decreased.Part II CONSTRUCTION OF FUSION PROTEIN AEMD AND LT70Objective:Construct the fusion protein Ag85B-ESAT6-Mtb8.4-RpFD(AEMD) and ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c(LT70) and make them stably express in E. coli BL-21 (DE3). To make them more express in the supernatant by codon optimization and joining linker and each antigen can be correctly folded without disturbing each other.Methods:1 The construction of Ag85B-ESAT6-Mtb8.4-RpFD:According to the Mycobacterium tuberculosis gene esat6, mtb8.4, rpfd from the GenBank, the primer was designed. After PCR, these genes were inserted into the expression vector pET30a (+) at the restriction sites Bgl Ⅱ, Sac Ⅰ, Hind Ⅲ in turn. After enzyme digestion, gene ag85b were inserted into the expression vector pET30a (+) at the restriction sites Nde Ⅰ and Bgl Ⅱ. The recombinant plasmid Ag85B-ESAT6-Mtb8.4- RpFD- pET- 30a (+) was constructed successfully, and then this plasmid was transformed into E. coli BL-21 (DE3) to express fusion protein Ag85B-ESAT6-Mtb8.4-RpFD. Depending on the front and the end sequence of ESAT6-Mtb8.4 which has achieved by ourselves, we designed a pair of primers. According to the precious experiences from our lab, the Ag85B is not expressed if it is inserted in front of the fusion protein. So when we designed this fusion protein, Ag85B was reformed by two ways, one is that the Ag85B sequence was adjusted, the other is that a linker GGGGS was implanted between Ag85B and ESAT6.2 The optimization of ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c:Based on the existing fusion protein in the laboratory, ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c, the Ag85B sequence was improved by both optimizing codon and joining a linker(GGGGS)3 at its both ends. When the Ag85B sequence, already designed, was synthetized by special factory, both ends’ restriction sites of the synthesis are the same as the original. After enzyme digestion, gene ag85b were inserted into the expression vector at the restriction sites EcoR Ⅰ and Bgl Ⅱ. The recombinant plasmid ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c-pET-30a (+) was constructed successfully, and then this plasmid was transformed into E. coli BL-21 (DE3) to express fusion protein ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c.Results:The fusion protein Ag85B-ESAT6-Mtb8.4-RpFD and ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c were successfully constructed, and they can stably express in E. coli BL-21 (DE3). Compared with the previous optimization, expression of both fusion proteins in the supernatant was increased.Conclusion:After the codon optimization and linker added, the expression of fusion protein Ag85B-ESAT6-Mtb8.4-RpFD and ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c was improved. Codon optimization and linker make each antigen be correctly folded so that more fusion protein could be expressed in supernatants.