Construction Tuberculosis Vaccine Fused Expression Ag85B-ESAT6 And Its Immunological Properties

Tuberculosis(TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis(MTB). It is estimated that one-third of the world population are infected with MTB, causing over 10 million new TB cases and 3 million deaths annually. In China, the number of TB patients was the second in the world, approximately 400 million people infected, over 5 million got sick, and 0.256 million died of TB annually. The only available vaccine against TB BCG, has been extensively evaluated and demonstrated a variable protective efficacy ranging from 0 to 80% in different field trials. Furthermore, due to following issues, such as the problem of TB multidrug-resistant (MDR) strains, co-infection with HIV, and increasing mobility of population, the word-wide situation of TB was deteriorating, which has created an urgent need for new vaccines to prevent TB .Ag85B and ESAT6 are both important secreted proteins in the short-term culture filtrate (ST-CF) of MTB, and involved in inducing multi-protective immunity. They are considered immunodominant and promising vaccine candidates as protective antigens for MTB challenge .In this study we hadcompared the levels of cell-mediated immune responses and protective efficacy by vaccines fused expression Ag85B and ESAT6 inducing subunit vaccine ,gene vaccine and recombinant BCG vaccine , and in order to search for a new effective TB vaccine .In this paper, the genes encoding Ag85B and ESAT6 of MTB mature proteins were amplified by polymerase chain reaction (PCR) from genome of MTB H37Rv strain, and inserted into cloning vector pGEM-T-easy for sequencing purpose. Genetic of Ag85B and ESAT6 were identical with that of Genbank reported, then digested by restriction endonuclease and cloned into expression vector pProEX HT. The recombinant plasmidsAz-pProEX HT-Ef and Ez-pProEX HT-Af were transformed into E.coli DH5, induced with IPTG, expressed fusion protein of Ag85B and ESAT6 with relative molecular mass(Mr) of 37 KD were confirmed by western blot analysis with mouse-specific monoclonal antibody against 6 X His. Fussed expression proteins were purified by Ni-NTA purification system.To test immunological properties of the fusion proteins, BALB/c mice were immunized three times at 2-week interval subcutaneously on their backs with fusion protein Ag85B-ESAT6 and ESAT6-Ag85B respectively, which were transferred to NC membranes beforehand. The titers of specific antibody in BALB/c mice sera immunized with Ag85B-ESAT6 is 1: 1000, and that of ESAT6-Ag85B is 1:5000. To evaluate cell-mediated immune response, stimulating index(SI) of the spleen lymphocytes in immunized mice were measured by MTT method, and the levels of IFN- Y secretion stimulated by antigen-specific were detected by indirect ELISA. The SI of experimental groups were significantly higher (which were 2.4 for Ag85B-ESAT6 and 2.6 for ESAT6-Ag85B respectively) than that of normal saline controls (0.9). IFN-r concentration in cultured supernatant of spleen lymphocytes from mice immunized with fusion proteins Ag85B-ESAT6 and ESAT6-Ag85B were 3.51+0.30ng/ml and 4.05 +0.4lng/ml respectively, significant greater than that ofcontrol group(0.5 + 0.25ng/ml, P<0.05), but lower than that of BCG immunized group (5.55 +0.31ng/ml) . To further evaluate protective immune response to MTB, BALB/c mice were intravenously infected with 105 CFU MTB H37Rv. Four weeks after the fina injection, compared with normal saline immunized mice, dramatic reduction in MTB replication was observed in the spleen(1.271og10 and 1.161og10 respectively, P<0.05) from BALB/c immunized with fusion proteins following a subsequent MTB H37Rv challenge, But the protection efficacy of mice immunized with Ag85B-ESAT6 or ESAT6-Ag85B was not good at that of BCG vaccination group(2.22 log10).The genes encoding Ag85B and ESAT6 were cloned into the corresponding sites of the eukaryotic expression vector pcDNA3, designated named Az-pcDNA3-Ef and Ez-pcDNA3-AF respectively, and then transfected into CHO cells by electroporation. The specific mRNA of the fused p...