白树毒素融合蛋白的筛选及其抗肿瘤作用和凋亡途径研究

Screening of a Gelonin Fusion Protein with High Cell-penetrating Efficiency and Its Anti-tumor Activity and Apoptosis Pathway

通过白树毒素(Gelonin)与多种穿膜肽(Cell-Penetrating Peptide,CPP)融合,获得一种抗肿瘤活性最高的Gelonin-HBP重组蛋白并研究其抑制肿瘤细胞增长的分子机制。以大肠杆菌BL21(DE3)重组表达及NI-NTA亲和纯化4种CPP(R9、TAT、HBD和HBP)与Gelonin融合的重组蛋白,通过超螺旋切割和体外翻译抑制实验检测其生物活性;激光共聚焦显微镜观察Gelonin重组蛋白穿膜效率、以MTT法检测其对肿瘤细胞的作用、并用流式细胞术及Western blot法观察其细胞凋亡的分子机制。结果显示,获得了保留Gelonin原有生物活性的4种Gelonin重组蛋白,连接CPP的Gelonin均能促进Gelonin的穿膜及抑杀肿瘤细胞效应,其中Gelonin-HBP活性最强,对于He La、B16、MCF-7和Hep G2四种肿瘤细胞比单独的Gelonin活性分别提升16.4倍、9.6倍、14.0倍和24.6倍;免疫印迹分析表明Gelonin-HBP诱导了肿瘤细胞Caspas 3、Caspas 8及Caspas 9的活性、Bax表达量显著增加,而Bcl-2的表达量显著降低。CPP促进了Gelonin对肿瘤细胞的抑杀和促凋亡作用,其中来源于人肝素结合表皮生长因子样生长因子的HBP连接Gelonin的抗肿瘤作用最强,其诱导肿瘤细胞凋亡的机制与线粒体和死亡受体介导的凋亡信号通路有关。

By fusing gelonin with a variety of cell penetrating peptides（CPP）,a recombinant protein gelonin-HBP with the highest antitumor activity was obtained and its molecular mechanism of inhibiting tumor cell growth was also studied. The recombinant protein of gelonin fused with four different CPPs（R9,TAT,HBD,and HBP）was expressed in Escherichia coli BL21（DE3）and then purified by NI-NTA affinity chromatography. Superhelical cleavage experiment and in vitro translation inhibition assay were used to detect the bioactivity of gelonin fusion protein. The translocation efficiency of gelonin recombinant protein was observed by laser scanning confocal microscopy. The cell growth inhibition effect of gelonin fusion protein on tumor cells was detected by MTT assay and the molecular mechanism of cell apoptosis was observed by flow cytometry and Western blot. The results showed that four gelonin recombinant proteins that retained the original biological activity of gelonin were obtained,and the fusion with a CPP promoted the cell penetration of gelonin,improved the inhibition effect of gelonin on tumor cells,and accelerated the apoptosis of these tumor cells. Compared to gelonin alone,the gelonin-HBP fusion protein increased its potency in 4 tumor cells of He La,B16,MCF-7 and Hep G2 by 16.4 times,9.6 times,14.0 times and 24.6 times,respectively. Western blotting analysis demonstrated that gelonin-HBP induced an enhancement in the activity of Caspas 3,Caspas 8 and Caspas 9 in tumor cells,thus the expression of Bax significantly increased,while the expression of Bcl-2 significantly decreased. Conclusively,CPP may enhance the anti-tumor effect and the apoptosis of gelonin on tumor cells;among them,HBP,a CPP derived from human heparin-binding epidermal growth factor-like growth factor,makes the gelonin-HBP have the strongest anti-tumor effect. The mechanism of inducing tumor cell apoptosis by this recombinant protein is related to mitochondrial and death receptor-mediated apoptosis signaling pathway.