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

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GFPCI／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Ni^2＋螯合柱亲和纯化融合蛋白GFPCI，SDS-PAGE检测融合基因的表达和纯化的融合蛋白GFPCI；纯化的融合蛋白GFPCI与HepG2细胞经体外孵育后在荧光显微镜下观察单链抗体Cl对肝癌细胞的靶向作用。结果：IPTG诱导表达后在荧光昆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GFPCI，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Ni^2＋亲和柱纯化获得较GFPCI重组融合蛋白，免疫荧光检测表明纯化的GFPCI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Cl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。

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

Objective: To express and purify of human scFv antibody(C1) against the asialoglycoprotein receptor fused to enhanced green fluorecsent protein, and observe its binding capacity to HepG2. Methods:The recombinant plasmid EGFPC1/pET-26b proved by DNA sequencing was transformed into E. coli BL21, and induced for fusion expression of EGFPC1with IPTG, the green fluorescence of E.coli BL21 harboring plasmid EGFPC1/pET-26b was observed under the fluorecsent microscope. The expressed EGFPC1 was purified with Ni2 chelating HiTrap HP column, and detected with SDS-PAGE. HepG2 was incubated with the recombinant EGFPC1, and the binding bioactivity was observed under the fluorecsent microscope. Results:The green fluorescence of E. coli BL21 harboring plasmid GFPS1/pET-26b was catched under the fluorecsent microscope. The recombinant GFPS1 protein was expressed in E.coli as inclusion body, rGFPS1 was prepared with Ni2 column purification. The result of immunofluorecsent detection verified that scFv C1 could specificially bind membrane of HepG2 cells. Conclusion:The purified EGFPC1 has strong binding capacity to the membrane of HepG2 using EGFP as a labeling protein, indicated the scFv C1 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.