| 引用本文: | 谭娇,陈应之,蔺婷婷,梁剑铭,杨永新,孙逊,黄永焯.肿瘤微环境双重响应性的智能型蛋白毒素给药系统的构建和表征[J].中国现代应用药学,2014,31(11):1297-1301. |
| TAN Jiao,CHEN Yingzhi,LIN Tingting,LIANG Jianming,YANG Yongxin,SUN Xun,HUANG Yongzhuo.Constructed and Characterization of Tumor Microenvironment-sensitive Drug Delivery System[J].Chin J Mod Appl Pharm(中国现代应用药学),2014,31(11):1297-1301. |
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| 摘要: |
| 目的 利用蛋白重组技术和PEG定点修饰技术，制备具有肿瘤微环境双重响应性的智能型蛋白毒素给药系统。方法 利用基因重组技术，在天花粉蛋白(trichosanthin，TCS)的C端引入天冬酰胺内肽酶(legumain)的底物天冬酰胺肽段和半胱氨酸残基，将所构建的突变体转化到大肠杆菌中表达目的蛋白并纯化。进一步将重组蛋白末端的半胱氨酸与具有巯基反应性的mPEG-Hz-Mal偶联合成TCS-Asn10-Hz-PEG，采用弱阳离子交换柱纯化TCS-Asn10-Hz-PEG，并在体外考察了TCS-Asn10-Hz-PEG的酸敏感性和酶敏感性。结果 成功制备、分离和纯化得到了TCS-Asn10-Cys，完成了mPEG-Hz-Mal与TCS-Asn10-Cys的定点偶联，得到智能型蛋白毒素给药系统TCS-Asn10-Hz-PEG。TCS-Asn10-Hz-PEG在体外pH 5.6的介质中和天冬酰胺内肽酶的作用下，能够水解或酶解释放出TCS，具有酸敏感特性和酶敏感特性。结论 本实验设计的蛋白毒素给药系统，具有酸敏感和酶敏感双重响应特性。 |
| 关键词: 天花粉蛋白 聚乙二醇修饰 肿瘤微环境 pH敏感 天冬酰胺内肽酶 |
| DOI： |
| 分类号:R284.1；R917.101 |
| 基金项目:国家自然科学基金(81422048, 81172996, 81373357) |
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| Constructed and Characterization of Tumor Microenvironment-sensitive Drug Delivery System |
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TAN Jiao1,2, CHEN Yingzhi2, LIN Tingting2, LIANG Jianming2, YANG Yongxin2, SUN Xun1, HUANG Yongzhuo2
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1.West China School of Pharmacy, Sichuan University, Chengdu 610041, China;2.Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
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| Abstract: |
| OBJECTIVE To achieve tumor microenvironment-sensitive drug delivery which was prepared by recombinant and PEGylation techniques. METHODS A substrate peptide sequence of asparaginyl endopeptidase (legumain) was introduced to the C-terminus of trichosanthin(TCS) by recombinant technique; the recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. TCS-Asn10-Cys recombinant protein was expressed and purified according a standard protocol. Further, the C-terminus thiol of the recombinant protein was conjugated to mPEG-Hz-Mal. The TCS-Asn10-Hz-PEG conjugate was purified using CM sepharose fast flow column. The pH-sensitivity and enzyme-sensitivity of TCS-Asn10-Hz-PEG in vitro were tested. RESULTS The TCS-Asn10-Cys fusion protein was successfully expressed and purified. The tumor microenvironment-drug delivery system of TCS-Asn10-Hz-PEG was prepared. In either the pH 5.6 medium or the presence of legumain, the PEG chain could be cleaved by hydrolysis or proteolysis from the TCS, of which the activity was recovered. CONCLUSION It is developed a dual-sensitive drug delivery system of TCS-Asn10-Hz-PEG that can be responsive to either acidic hydrolysis or legumain proteolysis. |
| Key words: trichosanthin PEGylation tumor microenvironment pH sensitive legumain |
