Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

【背景】卡介苗（Bacillus Calmette-Guérin, BCG）被视为极具应用前景的活细菌递送系统。然而，多款重组卡介苗（recombinant BCG, rBCG）疫苗的研发方案均未能取得进展，其主要制约因素为现有表达系统存在局限性。
【研究目的】本研究拟基于此前已证实具备良好稳定性的标准骨架载体，构建一系列搭载不同强度启动子的分枝杆菌载体。
【研究方法】以pLA71载体作为骨架，插入PAN、PαAg、PHsp60、PBlaF*及PL5等不同启动子，并以绿色荧光蛋白（green fluorescence protein, GFP）作为报告基因，构建分枝杆菌表达载体，以评估其相对启动强度、体外（巨噬细胞内）及体内稳定性。
【研究结果】不同载体的相对荧光检测结果显示，各启动子的强度呈递增趋势：在耻垢分枝杆菌（Mycobacterium smegmatis）与卡介苗中，PAN均为最弱的启动子；在卡介苗中，PBlaF*的启动强度高于PHsp60。在巨噬细胞系中检测到的相对荧光强度表明，PBlaF*与PHsp60的启动活性相当。在两种受试菌株中，均未能成功获得携带PL5的染色体外表达载体的转化菌株。
【主要结论】本研究成功构建了一系列表达水平各异、具备潜在稳定性的分枝杆菌载体，可用于重组卡介苗疫苗的研发工作。