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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: To establish a more broad-spectrum new targeted anthrax toxin delivery system and to evaluate its delivery efficiency by modifying the Protective Antigen of Anthrax toxin (PA) and lethal Factor (LF). Methods: Three kinds of recombinant natural anthrax toxin protective antigen PA and LFn-linker-Luc fusion gene of LFn-linker-Luc fused with LF N-terminal of anthrax toxin were constructed by genetic engineering. . The CCK-8 method was used to evaluate the cell viability after co-treatment with modified PA and LF. The modified PA and LFn-linker-Luc were co-incubated with tumor cells. The intracellular luciferase activity The effect of targeting tumor cells. Results: In vitro enzymatic hydrolysis experiments showed that the modified PA protein could be correctly digested into the target fragment. The reconstructed PA and LF cells could significantly reduce the cell viability, and LFn-linker-Luc Targeting PA efficiency and PA protein modification are related to their delivery efficiency. CONCLUSION: The designed and modified anthrax toxin drug delivery system can specifically target tumor cells and has a broader spectrum of effects, providing new ideas and methods for the development of new broad-spectrum anti-tumor drugs.