穿膜肽-β-葡萄糖苷酶偶联物的制备及活性鉴定  

Preparation and identification of CPPs-β-glucosidase conjugates

作　　者：王光文[1] 周洁[1] 王启辉[2] 刘云龙[1] 

机构地区：[1]广西医科大学第一附属医院,南宁530021 [2]广西医科大学基础医学院

出　　处：《山东医药》2013年第38期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81160314)

摘　　要：目的制备穿膜肽-β-葡萄糖苷酶偶联物并鉴定其穿膜活性和酶活性。方法采用异型双功能交联剂N-琥珀酰亚胺基-3-(2-吡啶二硫)-丙酸酯(SPDP)将穿膜肽—增强型绿色荧光蛋白融合蛋白(CPPs-EGFP)、β-葡萄糖苷酶进行共价交联,经Ni-NAT Superflow Cartridge、Sephadex G-75层析柱分离纯化。采用还原型和非还原型聚丙烯酰胺凝胶电泳(SDS-PAGE)对偶联物进行鉴定;偶联物与膀胱癌EJ细胞共同孵育,置于荧光显微镜下观察,鉴定其穿膜活性;采用β-葡萄糖苷酶测试盒(DBGD-100)鉴定其酶活性。结果偶联物SDS-PAGE非还原型电泳高分子量端可见明显条带,而还原型电泳分别在穿膜肽和β-葡萄糖苷酶分子量相应位置可见明显条带;在荧光显微镜下与偶联物共同孵育后的膀胱癌EJ细胞内可见明显绿色荧光;用β-葡萄糖苷酶测试盒(DBGD-100)测定,偶联物仍然保持良好的酶活性,约为同等浓度β-葡萄糖苷酶活性的80%。结论应用SPDP成功制备了穿膜肽-β-葡萄糖苷酶偶联物,并且偶联物保持良好穿膜活性和酶活性。该方法有助于解决酶—前药系统药物难以进入细胞内的问题。Objective To prepare cell-penetrating peptides （CPPs）-β-glucosidase conjugates and identify penetrating activity and enzymatic activity.Methods CPPs-enhanced green-fluorescent protein （EGFP） and β-glucosidase were crosslinked by N-succinimidyl-3-（2-pyridyldithio） propionate （SPDP） and purified by Ni-NAT Superflow Cartridge and Sephadex G-75 column chromatography.CPPs-β-glucosidase conjugates were tested by reduced and non-reduced SDSPAGE assay.CPPs-β-glucosidase conjugates and EJ cells were incubated together to detect the penetrating activity with fluorescence microscope and the enzymatic activity by DBGD-100.Results The non-reduced SDS-PAGE assay in detecting CPPs-β-glucosidase conjugates showed protein band at high molecular weight end,but the reduced SDS-PAGE assay showed protein bands corresponding to the sites of CPPs-EGFP and β-glucosidase.The EJ cells which were incubated with CPPs-β-glucosidase conjugates showed green fluorescent.After being tested by DBGD-100,the CPPs-β-glucosidase conjugates kept good enzymatic activity which might be 80％ of β-glucosidase activity at the same concentration.Conclusions CPPs-β-glucosidase conjugates can be prepared by the method of SPDP successfully,and the conjugates keep good penetrating and enzymatic activity.This method may be used to solve the problem that enzyme prodrug system cant easily enter the cell.

关 键 词：穿膜肽 Β-葡萄糖苷酶 偶联物 

分 类 号：R73-36[医药卫生—肿瘤]