组织基质金属蛋白酶抑制剂-2的表达纯化及活性鉴定  

Prokaryotic expression,purification and characterization of tissue inhibitor of metalloproteinase-2

作　　者：薛爱英 冯国兴 朱长春 樊赛军[1] Aiying Xue;Guoxing Feng;Changchun Zhu;Saijun Fan(Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,Institute of Radiation Medicine,Chinese Academy of Medical Sciences,Peking Union Medical College,Tianjin 300192,China)

机构地区：[1]中国医学科学院北京协和医学院放射医学研究所天津市放射医学与分子核医学重点实验室,天津300192

出　　处：《生物工程学报》2020年第12期2868-2876,共9页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.81703042,81730086)资助。

摘　　要：组织基质金属蛋白酶抑制剂-2(TIMP-2)抑制肿瘤迁移及侵袭。文中以人TIMP-2为研究对象,探索人TIMP-2蛋白的原核表达特征,并进行纯化及活性鉴定。以人肺癌A549细胞的总RNA反转录得到的cDNA为模板,克隆人TIMP-2基因,构建pET28a重组表达载体;经酶切检测和测序分析的重组表达载体pET28a-TIMP-2转入大肠杆菌Escherichia coli BL21(DE3)中,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并对表达条件进行优化。经镍亲和柱纯化后,用Western blotting法鉴定融合蛋白His-TIMP-2,并用明胶酶谱法检测融合蛋白的活性。研究发现融合蛋白His-TIMP-2在E.coli BL21(DE3)中以包涵体的形式存在;在一定范围内,IPTG浓度对His-TIMP-2的表达量没有显著影响;而在该表达系统中,诱导温度和时间是关键参数,His-TIMP-2的表达量随诱导温度升高而增加;纯化并复性后的融合蛋白His-TIMP-2能有效抑制人肺癌A549细胞表达的基质金属蛋白酶的活性。具有活性的融合蛋白的获得为后续深入研究人TIMP-2的功能及机制奠定基础,并对肿瘤治疗具有重要意义。Tissue inhibitor of metalloproteinases-2(TIMP-2)inhibits tumor migration and invasion.Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development.We collected human TIMP-2 protein through prokaryotic expression in vitro.We expressed,purified and characterized human TIMP-2 protein.First,the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells,and constructed to pET28a vector.The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3)after restriction endonuclease digestion and sequencing analysis.The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside(IPTG),and the expression conditions were optimized.After purification by nickel affinity column,the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography.The fusion protein His-TIMP-2 existed in the form of inclusion body in E.coli.In a certain range,the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2.But in this expression system,induction temperature and time were the key parameters,and the expression amount of His-TIMP-2 in E.coli increased with the increase of induction temperature.The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells.The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2,and is of great significance for tumor therapy.

关 键 词：基质金属蛋白酶抑制剂-2 重组表达载体 包涵体 纯化 基质金属蛋白酶 

分 类 号：R734.2[医药卫生—肿瘤] R730.5[医药卫生—临床医学]