目的为了建立对潜伏感染细菌的保护性免疫应答，本研究选择结核分枝杆菌休眠期、对数生长期最重要的保护性抗原HspX、Mpt64的190～198位的氨基酸多肽（CD8^＋T细胞表位）及Ag85B，构建融合蛋白Ag85B-Mpt64 190-198-HspX（AMH），并对其免疫原性进行研究。方法PCR分别扩增Ag85B、Mpt64 190-198-HspX基因，并依次插入pET-28a质粒中，在大肠杆菌BL21中表达纯化AMH蛋白。将该蛋白和佐剂二甲基三十六烷基铵和卡介苗多糖核酸（DDA＋BCG—PSN）混合构建亚单位疫苗，分别于1、4、7周皮下免疫C57BL／6小鼠3次，最后一次免疫5周后检测体液免疫与细胞免疫反应。结果该融合蛋白以包涵体形式能在大肠杆菌中稳定表达，经Ni—NTA层析柱纯化得到纯度较高的蛋白；构建的亚单位疫苗免疫动物能产生针对结核杆菌特定抗原（PPD、Ag85B、Mpt64 190-198和HspX）的特异性的细胞免疫和体液免疫应答，具有较强的免疫原性。结论融合蛋白AMH作为结核亚单位疫苗候选的融合蛋白抗原有必要进一步研究。

OBJECTIVE:To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN).METHODS:BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively.RESULTS:With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone.CONCLUSION:Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.

Objective To investigate the boosting efficiency of a subunit vaccine consisting of the fusion protein Ag85B-Mpt64190-198-Mth8.4 (AMM) , dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaceharide nucleic acid (BCG-PSN) on the primed inoculation with BCG. Methods The AMM subunit vaccine was composed of fusion protein AMM, adjuvant DDA and BCG-PSN. The first mouse experi-mental group was immunized with BCG first, then boosted with the AMM subanit vaccine in the 10th week. The second experimental group was boosted with the AMM subunit vaccine in the 8th week and the 10th week respectively with a two weeks interval after the primed with BCG. Two control groups were treated re-spectively with physiological saline alone and BCG alone. After the primed inoculation, ELISPOT and ELISA were used for the detection of the cell-mediated and humoral immune response in week 14 and week 22 re-spectively. Furthermore, the immunized mice were challenged with live BCG to mimic tuberculosis infection in the 22nd week after the primed inoculation. Subsequently the T cell typing and humoral response were de-tected by flow cytometry and ELISA, respectively. Results ( 1 ) The level of secreting IFN-γ: 14 weeks af-ter the primed inoculation,with the stimulation of the specific antigen-Ag85B, the number of cells secreting IFN-γ in the second experimental group (135±14) was more than BCG alone immunized group (19±16), t = 10. 98, P < 0.01. In the 22nd week, the number of cells secreting IFN-γ in the second experimental group (208±11) was still more than BCG alone group (57±18), t =6.43, P <0.01. (2) The level of humoral immune response: the IgG1 antibody titer in the second experimental group was obviously higher than that in the first experimental group. However, the ratio of IgG2a to IgG1, as the index reflecting the Thl-type immune response, in the experimental group 2 was lower than that in the experimental group 1. (3) The contents of CD4+ CD25+ T cells after challenged with live BCG strain: the first and the second ex-perimental groups were both higher than the BCG alone group (t1 = 3.08, t2 = 3.16, P < 0.05 ). Conclu-sion Boosting the BCG-pfimed mice with tuberculosis AMM subunit vaccine twice can induce higher level of cell-mediated and humoral immune response than BCG alone, which could activate the regulative immune response at the same time.

Objective To construct protective immunity to Mycobncterium tuberculosis latent infection, a novel fusion protein consisting of HspX, the 190 to 198 peptide of Mpt64 and Ag85B, which were confirmed to be the effective protective antigens mainly expressed in the dormancy and exponential phase of growth, was constructed and its immunogenicity was investigated. Methods Ag85B and Mpt64190-198-HspX sequences were amplified by PCR and cloned into plasmids pET-28a. The fusion protein, Ag85BMpt64190-198-HspX (AMH) was expressed in E. coli BL21 and purified with Ni-NTA resins. C57BL/6 mice were immunized three times at 2-week intervals subcutaneously with AMH formulated with the adjuvant composed of dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCGPSN). Humoral and cell-mediated immunity responses were analyzed at five weeks after the last injection. Results AMH was expressed stably in E. coli and could be purified well by Ni-NTA affinity chromatography. C57BL/6 mice immunized with AMH subunit vaccine generated specific cellular and humoral immunologic response to the stimulation of Ag85B, Mpt64190-198 and HspX. Conclusion It suggested that AMH was a promising candidate antigen of tuberculosis subunit vaccine.

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64(190-198)-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA-BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-gamma was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64190–198-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA–BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-γ was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.