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  

Constructed and Characterization of Tumor Microenvironment-sensitive Drug Delivery System

作　　者：谭娇[1,2] 陈应之 蔺婷婷[2] 梁剑铭[2] 杨永新[2] 孙逊[1] 黄永焯[2] 

机构地区：[1]四川大学华西药学院,成都610041 [2]中科院上海药物所,上海201203

出　　处：《中国现代应用药学》2014年第11期1297-1301,共5页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金(81422048;81172996;81373357)

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m PEG-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m PEG-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p H 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。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 m PEG-Hz-Mal. The TCS-Asn10-Hz-PEG conjugate was purified using CM sepharose fast flow column. The p H-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 p H 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.

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 

分 类 号：R963[医药卫生—微生物与生化药学]