摘要
将核糖体失活蛋白类鼻疽伯克霍尔德菌致死因子1(BLF1)与细胞穿膜肽(CPP)HBP融合表达并与商陆皂苷甲(EsA)联合使用,提高BLF1重组蛋白抑制肿瘤细胞生长的活性。通过原核表达及NI-NTA亲和层析纯化BLF1、BLF1-HBP融合蛋白,以HepG2、MCF-7、A549和HeLa细胞为检测模型,MTT法检测BLF1重组蛋白对肿瘤细胞的毒性,激光共聚焦显微镜观察重组蛋白进入细胞效率,流式细胞术分析抗肿瘤效应。结果显示,穿膜肽HBP可有效提高BLF1对HepG2、MCF-7、A549和HeLa四种肿瘤细胞的生长抑制作用,其中对HeLa细胞的药效增强效果最显著,可达47.5倍;皂苷EsA的使用进一步显著提高了BLF1-HBP对上述4种肿瘤细胞生长抑制活性,且对MCF-7细胞的IC50值从6840nmol/L降至0.57nmol/L。激光共聚焦观察揭示EsA可有效促进BLF1重组蛋白进入肿瘤细胞效率,流式结果分析表明EsA可大大强化BLF1-HBP诱导肿瘤细胞凋亡的能力。将BLF1与穿膜肽HBP融合表达并在EsA存在下,可显著提升BLF1对肿瘤细胞的毒性,强化其诱导肿瘤细胞凋亡的能力。
A ribosome-inactivated protein named as Burkholderia lethal factor 1(BLF1)was fused with the cell penetrating peptide (CPP)HBP and co-administered with esculentoside A(EsA)to enhance the antitumor effect of the recombinant protein BLF1-HBP. Both the recombinant protein BLF1 and BLF1-HBP were expressed in Escherichia coli BL21(DE3)and then purifed with NI-NTA. The inhibitory effects of the two recombinant proteins were tested and compared in tumor cell line HepG2,MCF-7,A549 and HeLa with the MTT assay. The transmembrane efficiency of the recombinant proteins was observed by laser confocal microscopy and the anti-tumor effect was analyzed with flow cytometry. The result showed that CPP HBP effectively increased the inhibitory effect of BLF1 in all 4 tested tumor cell lines,and the most significant inhibitory effect was observed in HeLa cells,which was increased by 47.5 times. When BLF1-HBP was co-administered with EsA, the antitumor activity of BLF1-HBP was further improved and the IC 50 value in MCF-7 cells decreased from 6 840 nmol/L to 0.57 nmol/L. Laser confocal observation revealed that EsA effectively promoted the translocation efficiency of BLF1 recombinant protein. Flow cytometry analysis showed that EsA greatly enhanced the apoptosis effect of tumor cells induced by BLF1-HBP. Therefore,the fusion of BLF1 and HBP and co- administration with EsA may significantly increase the antitumor effect of BLF1,and this enhancement is due to the induction of more tumor cell apoptosis.
作者
刘洋
曹雪玮
卢美雅
王富军
赵健
LIU Yang;CAO Xue-wei;LU Mei-ya;WANG Fu-jun;ZHAO Jian(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237;Zhejiang Fonow Medicine Co. Ltd.,Dongyang 322100;Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203)
出处
《生物技术通报》
CAS
CSCD
北大核心
2019年第8期146-154,共9页
Biotechnology Bulletin
基金
国家自然科学基金项目(81571795)