靶向递送人颗粒溶素与小鼠单链IL-12真核共表达质粒耻垢分枝杆菌的构建及鉴定

Construction and identification of a Mycobacterium smegmatis strain to targetedly deliver eukaryotic coexpression plasmid encoding human granulysin and murine single chain IL-12

目的:构建携带编码人天然颗粒溶素(GLS)和小鼠单链IL-12基因的真核共表达载体pZM03,并构建可将其靶向递送入巨噬细胞中进行表达的重组耻垢分枝杆菌菌苗.方法:将人GLS,小鼠单链IL-12基因和分枝杆菌复制子OriM同时克隆进多启动子真核共表达载体pBudCE4.1中,得到穿梭质粒pZM03.以pZM03电转化耻垢分枝杆菌mc2-155,通过PCR方法检测重组菌中携带的真核共表达质粒;通过与小鼠巨噬细胞株RAW264.7共孵育检测重组菌的靶向性.结果:成功得到了可靶向递送GLS/IL-12真核共表达质粒pZM03进入巨噬细胞的新型重组菌.结论:利用耻垢分枝杆菌作为减毒生物体载体向巨噬细胞中靶向递送真核表达质粒是可能的.

AIM： To construct a recombinant Mycobacterium smegmatis strain that can targetedly deliver the eukaryotic coexpression plasmid pZM03 encoding human granulysin （GLS） and murine single chain IL-12 into macrophages. METHODS： Coding sequences of human granulysin, murine single chain IL-12 and OriM were simultaneously cloned into pBudCE4. 1, which had multiple promoters to form the shuttle plasmid pBudCE4. 1/GLS/ IL12/OriM （simply named pZM03 ）. The shuttle plasmid was transformed into mc2-155 and identified from the recombinants by PCR. The targeting property of the recombinant was detected by co-incubating the strains with murine macrophage RAW264. 7. RESULTS： The new recombinant strain that could targetedly deliver pZM03 into macrophages was successfully constructed. CONCLUSION： It is feasible to targetedly deliver eukaryotic expression plasmids into macrophages by using Mycobacterium smegmatis as an attenuated biological vector.