GLS/IL-12重组BCG的构建及其对小鼠结核病疗效的研究

GLS/IL-12 modified BCG as a novel antituberculosis immunotherapy

目的构建携带编码人天然颗粒溶素(GLS)和小鼠IL-12基因质粒(pZM03)的重组卡介苗(BCG),并观察其对结核分枝杆菌感染的疗效与机制。方法将分枝杆菌复制子OriM克隆进已含GLS和IL-12的多启动子真核共表达载体pBudCE4.1中(pZM02),得到穿梭质粒pZM03,以pZM03电转化BCG获得重组BCG。结核分枝杆菌H37Rv感染Balb/c小鼠4周后分别用生理盐水、BCG、pZM03和重组BCG治疗,于第一次治疗后3个月,处死小鼠,检测器官荷菌量、脾淋巴细胞IFN-γ和TNF-α的分泌水平、血清IL-12和肺、脾组织中GLS表达,同时观察小鼠肺、脾组织病理改变情况。结果重组BCG治疗组肺、脾组织荷菌量比生理盐水组、BCG组和质粒组显著降低。重组BCG组和pZM03组经PPD刺激后IFN-γ和TNF-α分泌水平明显高于BCG组和生理盐水组。重组BCG组血清IL-12水平明显高于生理盐水组,但与pZM03质粒组和BCG载体组无明显差异。用免疫组化检测到小鼠肺及脾组织中GLS的表达。生理盐水和BCG组肺组织病理改变以渗出为主,病变广泛,增生改变不明显;重组BCG病变范围局限,并有大量类上皮细胞、泡沫细胞等细胞出现。结论成功构建携带GLS和IL-12的重组BCG;重组BCG对小鼠结核病有一定免疫治疗作用,系增强宿主Th1型免疫应答和抗菌活性有关。

In this study, an eukaryotic coexpression plasmid encoding human granulysin and murine IL-12 was constructed and the effects and mechanisms of recombinant BCG encoding human granulysin and murine IL-12 on murine M. tuberculosis infection was studied. Coding sequences of human granulysin, murine single chain IL-12 and （）riM were simultaneously cloned into pBudCE4.1 which has multiple promoters to form the shuttle-plasmid pBudCE4.1/GLS/IL12/OriM （pZM03）. The shut- tle-plasmid was transformed into BCG and identified by PCR. Balb/c mice infected with M. tuberculosis were treated with normal saline, BCG, pZM03, recombinant BCG respectively. The recombinant strain delivering pZM03 into mice was successfully constructed. The recombinant BCG group and pZM03 group showed significantly reduced number of colony forming units（log CFU/g） compared with the control and BCG group. The expression of GLS in tissue and the increased of IL-12 in serum were found in recombinant BCG group. IFN-7 and TNF-α released from spleen lymphocytes stimulated with PPD in recombinant BCG group and pZM03 group was higher than that of other groups, the difference between recombinant BCG group and pZM03 group was not significant. Pathological changes in lungs of the recombinant BCG group were localized, while those in the control group were extensive. The pathological changes were not found in the spleens of all groups. It is evident that recombinant BCG shows immunotherapeutic effects, which may be associated with a switch to Thl response and the antibacterial activity of GLS.