一种用于肿瘤药物治疗的新型人源性穿膜肽的优化及其应用 <sup>*</sup>

doi:10.13523/j.cb.20180706

中国生物工程杂志

2018, Vol. 38

Issue (7): 40-49 DOI: 10.13523/j.cb.20180706

技术与方法

一种用于肿瘤药物治疗的新型人源性穿膜肽的优化及其应用 ^*

李思¹,翟逸舟¹,陆玉婷²,王富军^2,³,赵健^1,^**(

)

1 华东理工大学生物反应器工程重点实验室上海 200237
2 浙江日升昌药业有限公司东阳 322100
3 上海中医药大学中药研究所上海 201203

The Optimization of A Novel Human-derived Cell-penetrating Peptide Used for Anti-cancer Treatment

Si LI¹,Yi-zhou ZHAI¹,Yu-ting LU²,Fu-jun WANG^2,³,Jian ZHAO^1,^**(

)

1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, Chain
2 Zhejiang Reachall Pharmaceutical Co. Ltd, Dongyang 322100,Chain
3 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203,Chain

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摘要：

细胞穿膜肽(cell-penetrating peptide,CPP)作为一种体内大分子药物跨膜运输载体被广泛研究和应用。中期因子Midkine(MK)是人体的一种带有肝素结合域(heparin-binding domain,HBD)的生长因子。报道了MK中HBD的部分富含碱性氨基酸的基因序列(命名为MK-S0)与绿色荧光蛋白(EGFP)基因融合表达后,能将EGFP有效地转运入胞内,且其穿膜转运效率高于经典穿膜肽Tat。将MK-S0序列进一步突变优化改造得到的midkine-mutant Δ4(MK-Δ4),其穿膜效率比天然序列来源的MK-S0提高16倍以上,且MK-Δ4的穿膜转运作用适用于多种肿瘤细胞。穿膜机制分析研究结果显示,MK-Δ4可与细胞表面硫酸乙酰肝素结合,随之以巨胞饮形式内吞入胞。采用MTT方法检测的细胞生长抑制试验结果显示,连接有MK-Δ4的苦瓜来源的核糖体失活蛋白MAP30比单独的MAP30对HeLa肿瘤细胞的药效可提升5.8倍,大大提高了这种药物蛋白抑杀肿瘤细胞的效果。由此表明,源于MK的这种经过突变改造的MK-Δ4,可作为一种新型高效的细胞穿膜肽,将药物蛋白有效运输到细胞内发挥抗肿瘤效应。

关键词： 中期因子; 肝素结合域; 细胞穿膜肽; 药物运输; 抗肿瘤药物

Abstract:

Cell-penetrating peptides (CPPs) have been widely used in decades for its ability to carry many macromolecular drugs across cell-membrane to exert their effects. Midkine (MK) is a heparin-binding growth factor with a heparin-binding domain (HBD). The HBD in MK that is rich in basic amino acids (MK-S0) was fused with enhanced green fluorescence protein (EGFP) and then it was found that MK-S0 could deliver EGFP into cells, and its transportation capacity is much higher that classical CPPs (such as Tat). After the sequence optimization on MK-S0, MK-Δ4 whose trans-membrane ability was increased about 16-fold than MK-S0 was obtained. The trans-membrane ability of MK-Δ4 was also suitable for a variety of tumor cells. The further investigation of endocytic pathways on MK-Δ4 was shown that MK-Δ4 penetrates cell-membrane through interacting with heparin sulfate on the cell surface and then via macropinocytosis. The results of cell growth inhibition by MTT method showed that MK-Δ4 could enhance the inhibitory effect of a ribosome-inactivating protein-MAP30 about 5.8-fold in HeLa cells which is significantly enhance the anti-tumor activity of MAP30. It was suggested that MK-Δ4 optimized from heparin-binding domain MK is a novel human-derived CPP with high efficiency, and is a new drug vector for anti-tumor therapy.

Key words: Midkine Heparin-binding domain Cell-penetrating peptide Drug delivery Anti-tumor drug

收稿日期: 2018-03-20 出版日期: 2018-08-13

ZTFLH:

Q71

基金资助: 国家自然科学基金(81571795)

通讯作者: 赵健 E-mail: zhaojian@ecust.edu.cn

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引用本文:

李思,翟逸舟,陆玉婷,王富军,赵健. 一种用于肿瘤药物治疗的新型人源性穿膜肽的优化及其应用 ^*[J]. 中国生物工程杂志, 2018, 38(7): 40-49.

Si LI,Yi-zhou ZHAI,Yu-ting LU,Fu-jun WANG,Jian ZHAO. The Optimization of A Novel Human-derived Cell-penetrating Peptide Used for Anti-cancer Treatment. China Biotechnology, 2018, 38(7): 40-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180706 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/40

图1 纯化的重组蛋白EGFP-MK-S0进行SDS-PAGE分析

图2 EGFP-MK-S0及多种CPPs的荧光检测及流式细胞术检测

图3 EGFP-MK-S0突变体的穿膜效率分析

表1 MK-S0的突变体序列

图4 纯化的重组蛋白EGFP-MK-Δ4的SDS-PAGE分析

图5 EGFP-MK-Δ4的穿膜细胞谱

图6 各种内吞途经抑制剂对EGFP-MK-Δ4穿膜效率的影响

图7 EGFP、EGFP-MK-Δ4对HeLa的促生长效应

图8 纯化的重组蛋白MAP30-MK-Δ4的 SDS-PAGE分析

图9 重组蛋白MAP30-MK-Δ4对不同肿瘤细胞存活率的影响

表2 MAP30融合蛋白对不同肿瘤细胞的半抑制浓度 (IC50)

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