去唾液酸糖蛋白受体单链抗体与绿色荧光蛋白的融合表达及其靶向性观察  

Expression and bioactivity detection of scFv C1 against asialoglycoprotein receptor with enhanced green fluorecsent protein

作　　者：卢志贤[1] 陈江[1] 程华莉[1] 潘宇红[1] 曹利民[1] 

机构地区：[1]南通大学附属无锡三院检验科,江苏无锡214041

出　　处：《南京医科大学学报（自然科学版）》2006年第12期1192-1195,F0003,共5页Journal of Nanjing Medical University(Natural Sciences)

摘　　要：目的:表达和纯化去唾液酸糖蛋白受体单链抗体C1与增强型绿色荧光蛋白(EGFP)的融合蛋白,体外观察其肝癌细胞的特异性结合能力。方法:通过扩增构建成含C1与绿色荧光蛋白(GFP)融合基因的原核表达质粒GFPC1/pET-26b;测序验证后转化大肠杆菌BL21后以IPTG诱导表达,荧光显微镜下观察融合基因中GFP的表达情况;用Ni2+螯合柱亲和纯化融合蛋白GFPC1,SDS-PAGE检测融合基因的表达和纯化的融合蛋白GFPC1;纯化的融合蛋白GFPC1与HepG2细胞经体外孵育后在荧光显微镜下观察单链抗体C1对肝癌细胞的靶向作用。结果:IPTG诱导表达后在荧光显微镜下可以观察到大肠杆菌发出特异性的绿色荧光;SDS-PAGE显示表达的融合蛋白GFPC1,与预期的大小相一致,以包涵体的形式存在;通过变性条件下Ni2+亲和柱纯化获得较GFPC1重组融合蛋白,免疫荧光检测表明纯化的GFPC1可与HepG2细胞膜特异结合。结论:利用GFP作为标记分子,观察到去唾液酸糖蛋白受体单链抗体C1与HepG2细胞膜较强的结合能力,为应用C1对肝癌进行靶向生物治疗奠定基础;构建成功的GFP/pET-26b原核表达系统为其他靶分子的研究提供了一个有力的工具。Objective： Tu express and purify of human scFv antibody （CI） against the asialoglycoprotein receptor fused to enhanced green fluorecsent protein, and observe its binding capacity to HepG2. Methods ：The recombinant plasmid EGFPCI/pET-26b proved by DNA sequencing was transformed into E. coil BL21, and induced for fusion expression of EGFPCIwith IPTG, the green fluorescence of E.coli BL21 harboring plasmid EGFPCl/pET-26b was observed under the fluorecsent microscope. The expressed EGFPCl was purified with Ni^2＋ chelating HiTrap HP column, and detected with SDS-PAGE, HepG2 was incubated with the recombinant EGFPC1, and the binding bloactivity was observed under the fluorecsent microscope. Results：The green fluorescence of E. coli BL21 harboring plasmid GFPSI/pET-26b was catched under the fluorecsent microscope. The recombinant GFPSI protein was expressed in E.coli as inclusion body, rGFPSI was prepared with Ni^2＋ column purification. The result of immunofluorecsent detection verified that scFv CI could specificially bind membrane of HepG2 cells. Conclusion：The purified EGFPCI has strong binding capacity to the membrane of HepG2 using EGFP as a labeling protein, indicated the scFv CI has a potential value as a targeting molecule for biological therapy of hepatoma; The constructed EGFP/pET-26b can also be used for research of other targeting molecule.

关 键 词：肝癌 单链抗体 增强型绿色荧光蛋白 融合表达 

分 类 号：R392.1[医药卫生—免疫学]