应用改构的炭疽毒素作为靶向肿瘤细胞的药物递送系统及其效果评价

Construction and Evaluation of the Mutated Anthrax Toxin Proteins as Drug Deliver System for Targeting Tumor Cells

目的:通过改造炭疽毒素保护性抗原Protective Antigen(PA)及致死因子Lethal Factor(LF),尝试建立更加广谱的新型炭疽毒素靶向给药系统并对其递送效率进行定量评价。方法:采用基因工程手段,分别构建了3种改构的天然炭疽毒素保护性抗原PA及炭疽毒素的LF N端融合海肾荧光素酶(Luciferase)的LFn-linker-Luc的大肠杆菌重组表达体系。利用CCK-8法评价改构PA和LF共同作用肿瘤细胞后的细胞存活率;利用改构PA和LFn-linker-Luc与肿瘤细胞共孵育,通过测定细胞内荧光素酶活性,评价改构PA靶向肿瘤细胞的效果。结果:体外酶解实验证明构建的改构PA蛋白能够被正确地酶解成目的大小的片段;改构PA和LF共同作用肿瘤细胞能够显著降低细胞存活率;利用LFn-linker-Luc能够评价改构PA的靶向效率,PA蛋白的改构方式与其递送效率相关。结论:设计并改构的炭疽毒素药物递送系统,能够实现特异性靶向肿瘤细胞的效果,并具有更广谱的作用效果,为研制新型广谱抗肿瘤药物提供了新的思路和方法。

Objective： The anthrax toxin proteins constitute a unique membrane trans]ocation system which provide an attractive model system for the development of reagents for cell biology and therapeutics. The mutated anthrax toxin proteins PA and LF were constructed to target to tumor cells. Methods： The recombinant Rosseta （DE3） which harboring three kinds of mutated PA and LF were constructed separately. The cell viability after incubated with mutated PA and LF was detected by cytotoxicity assay. The ability of the mutated PA translocation system targeting to tumor cells was measured by the amount of the luciferase of intra-cellular. Results： The mutated PA can be cleaved by matrix metalloproteinases or uPA in vitro correctly. When the tumor cell incubated with the mutated PA and LF,the cell viability was decreased remarkably. The amount of the luciferase of intracellular can correctly reflex the ability of the mutated PA translocation system targeting to tumor cells. Conclusions： These mutated PA proteins showed natural biological activities and had the ability of targeting to tumor ceils. These results provide a new insight for the study of the antitumor drugs.