肺癌靶向通用载体hu3D3／cSA融合蛋白的制备和活性分析  

Preparation and activity analysis of hu3D3/core-streptavidin fusion protein as a universal carrier targeting to lung cancer

作　　者：王勇军[1] 颜江华[2] 王阶平[1] 王臻[1] 黄志平[1] 陈彩霞[1] 

机构地区：[1]厦门大学生命科学学院,361005 [2]厦门大学医学院抗癌研究中心,361005

出　　处：《中华微生物学和免疫学杂志》2008年第2期114-118,共5页Chinese Journal of Microbiology and Immunology

基　　金：福建省自然科学基金（00410004）

摘　　要：目的制备一种新型的肺癌靶向通用载体hu3D3与核心链霉亲和素（core-streptavidin，cSA）的融合蛋白hu3D3／cSA，并鉴定其活性。方法PCR扩增cSA基因，克隆至质粒hu3D3／pET-22b（＋）而获得表达载体hu3D3／cSA／pET-22b（＋），并在E.coli BL21（DE3）中表达，镍亲和层析柱纯化hu3D3／cSA融合蛋白，采用TEA缓冲液进行复性。SDS-PAGE和Westem blot鉴定融合蛋白四聚体的形成。FTTC标记hu3D3／cSA，通过荧光显微镜分析hu3D3组分的抗原反应性和特异性，流式细胞术比较hu3D3／cSA和hu3D3与靶点的结合能力；ELISA和Westem blot鉴定cSA组分结合生物素（biotin）的能力。结果获得序列正确的hu3D3／cSA／pET-22b（＋）重组子，融合基因在E.coli BL21（DE3）中主要以包涵体的形式表达。纯化复性后的融合蛋白能够形成四聚体复合物，保留了cSA结合生物素的活性，hu3D3组分能与肺癌细胞表面抗原结合，且结合能力与单体hu3D3相比有明显增强。结论成功制备了具有结合靶抗原和生物素活性的hu3D3／cSA融合蛋白，同时改善了hu3D3组分结合其靶点的亲合力（avidity）。因此，hu3D3／cSA可以作为一个通用的肺癌靶向载体，与多种生物素化的抗肿瘤药物或试剂联用，有望为多药物联合靶向治疗肺癌提供一种可行的方法。Objective To prepare a novel fusion protein of hu3D3 and core-streptavidin（hu3D3/ cSA） as a universal carrier targeting to lung cancer, and analyze its activities. Methods cSA gene was acquired by PCR, and inserted into plasmid hu3D3/pET-22b（ ＋ ） to construct a recombinant plasmid hu3D3/ cSA/pET-22b（ ＋ ）. The fusion gene was expressed in E. coli BL21 （DE3）. The fusion protein was purified through nickel-affinity chromatography column and renatured using TEA buffer. The tetrameric hu3D3/cSA complex was analyzed by SDS-PAGE and Western blot. The hu3D3/cSA protein was labeled with FITC, then its antigen-binding activity was analyzed using fluorescence microscopy, and the functional affinity of hu3D3/cSA and hu3D3 were analyzed and compared by flow cytometry. The biotin-binding activity of hu3D3/cSA was tested by ELISA and Western blot. Results The recombinant plasmid hu3D3/cSA/pET- 22b（ ＋ ） with correct sequence was obtained. The fusion protein was found after expression in E. coli BL21 （DE3） mainly in the form of inclusion body. After being purified and refolded, tetrameric complex was formed. The purified tetrameric hu3D3/cSA complex retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety; furthermore, the avidity of the hu3D3/cSA to its target antigen increased by about 14 times as compared with that of monomeric hu3D3. Condusion The fusion protein hu3D3/cSA with both antigen- and biotin-binding activity is successfully prepared, and the avidity of hu3D3 moiety to 3D3 antigen is enhanced. Consequently, hu3D3/cSA could be a universal carrier targeting to lung cancer, and could be an alternative, convenient method to realize target therapy to lung cancer by the combination of multiple biotinylated anti-tumor drugs or agents.

关 键 词：通用载体 单域抗体 核心链酶亲和素 肺癌 

分 类 号：R686[医药卫生—骨科学]