人源化抗人肺癌二价和四价VH单域抗体的制备及活性分析  

Preparation and activity analysis of divalent and tetravalent humanized single-domain antibody against human hong cancer

作　　者：王阶平[1] 颜江华[1] 张长弓[1] 王勇军[1] 庄国洪[1] 

机构地区：[1]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》2007年第2期176-179,共4页Chinese Journal of Microbiology and Immunology

基　　金：福建省自然科学基金项目（No.C0410004）

摘　　要：目的 为提高人源化的抗人肺癌单域抗体hu3D3va的功能性亲和力，制备其二价和四价的抗体分子。方法 重组PCR分别将SV5-Cys短肽和p53四聚化结构域基因与hu3IY3Va基因融合，而获得同源二聚体dihu3D3vH和同源四聚体tehu3D3Va基因。将两种基因克隆至pET-22b（＋）表达载体，并分别在E-coliB121（DE3）中表达。表达产物通过N^2＋亲和层析柱纯化。用FITC分别标记hu31Y3VH、dihu 31Y3VH和tehu3D3VH三种抗体分子，通过免疫荧光实验分析其抗原反应性和特异性，并利用流式细胞术比较分析其功能性亲和力。结果 两种基因均以单体形式并主要以包涵体的形式表达，纯化并复性后，其蛋白溶液中分别存在约50％的二聚体和70％的四聚体。dihu3D3VH和tehu3D3VH均保留了hu3IY3VH的抗原反应性和特异性；且与hu3D3VH相比，其功能性亲和力分别提高了约60％和160％。结论 采用增加结合价的策略，有效地改善了hu3D3VH的功能性亲和力。Objective To improve the functional affinity of humanized single-domain antibody hu3D3V. against human lung cancer. Methods Divalent and tetmvalent variants of hu3D3VH were prepared. Genes of homogeneous dimers dihu3D3VH and tetramers tehu3D3VH were constructed by fusing the SVS-Cys and the p.53 tetramerization domain gene to the hu3D3VH gene using recombinant PCR technique. The two genes were cloned into expression vector pET-22b（ ＋ ） and expressed in E. coli BI21（DE3）. The expressed products were purified through Ni^2＋ atSnity chromatographic column. The hu3D3V,, dihu3D3V, and tehu3D3V, proteins were labeled with FITC. Then their reactivifies and specitlcities were analyzed by fluoroimmunoassay, and their functional affinities were analyzed and compared by flow cytometry. Results The two genes were both expressed as monomers and mainly as inclusion bodies. There were about 50% dimers and 70% tetramers in the dihu3D3VH and tehu3D3VH protein resolution after purification and renaturation. The dihu3D3VH and tehu3D3VH still keep the reactivity and specificity of hu3D3VH ; and their functional affinities increased from 60% to 160%, comparing with hu3D3VH. Conclusion The functional affinity of hu3D3VH was greatly improved by increasing its valency.

关 键 词：单域抗体 同源聚合体 结合价 功能性亲和力 

分 类 号：R686[医药卫生—骨科学]