穿膜肽凋亡素融合蛋白诱导黑色素瘤细胞凋亡的实验研究

Melanoma Cells Apoptosis Induced by Novel Cell-Penetrating Peptide and Apoptin Fusion Protein

目的探讨新型细胞穿膜肽h PP10和凋亡素Apoptin融合蛋白介导小鼠黑色素瘤细胞B16凋亡的效应。方法构建p ET15b-h PP10-Apoptin、p ET15b-Apoptin原核表达质粒,经IPTG诱导和NI-NTA树脂纯化后得到融合蛋白;运用TUNEL法体外检测h PP10-Apoptin诱导B16细胞的凋亡效应;制备荷瘤小鼠模型,并在体内检测h PP10-Apoptin融合蛋白的抗肿瘤能力。结果融合蛋白h PP10-Apoptin经Western blot鉴定正确,且此融合蛋白能在体内外有效诱导B16黑色素瘤细胞凋亡。结论新型细胞穿膜肽h PP10可有效携带Apoptin穿透细胞膜并发挥抗肿瘤作用。

OBJECTIVE To investigate apoptosis induction ability of hPP10-Apoptin fusion protein to mouse B16 melanoma cell. METHODS pET15b-hPP10-Apoptin expression plasmid was constructed, and E. coli BL21 （DE3） was transformed with the plasmid, then the yielded hPP10-Apoptin fusion protein was purified by Ni-NTA His-Bind Resin and confirmed by Western blotting assay. Melanoma cell apoptosis induced by hPP10-Apoptin fusion protein was analyzed by TUNEL assay, and the antitumor effect was examined in melanoma cell-bear mouse model. RESULTS hPP10-Apoptin fusion protein was highly expressed in BL21 cells, Western blotting analysis result showed that fusion protein was expressed correctly. The fusion protein can induce melanoma cell apoptosis in vivo and in vitro. CONCLUSION The results confirm that hPP10-Apoptin fusion protein could penetrate into melanoma cell and also has antitumor effect.