EGFRvIII胞外区基因重组腺病毒的构建及单域抗体的制备

Construction of recombinant adenovirus expressing EGFRvIII extracellular domain gene and preparation of single domain antibody

摘要:

本研究旨在构建能够表达人表皮生长因子EGFRvIII胞外区基因的重组腺病毒，并通过免疫骆驼构建噬菌体单域抗体库，筛选和制备EGFRvIII胞外区特异性单域抗体并对其进行鉴定。从人前列腺癌细胞系PC-3细胞中提取总RNA，反转录为cDNA，以cDNA为模板扩增EGFRvIII胞外区基因，并连接pAdTrack-CMV质粒载体，转化含有pAdEasy-1的大肠杆菌(Escherichia coli) BJ5183感受态细胞，获得同源重组质粒转染HEK293A细胞，得到表达EGFRvIII胞外区蛋白的重组腺病毒。利用重组腺病毒免疫双峰驼，构建EGFRvIII胞外区特异性噬菌体单域抗体库，并以EGFRvIII蛋白为筛选抗原，对其进行筛选，对筛选得到的单域抗体进行诱导表达、纯化及鉴定。结果表明获得了表达EGFRvIII胞外区基因的重组腺病毒。构建得到的EGFRvIII特异性噬菌体单域抗体库的库容为1.4×10⁹；经过3轮富集和筛选，通过噬菌体ELISA筛选出31个与EGFRvIII胞外区蛋白结合的阳性克隆，并对OD₄₅₀值较高的重组单域抗体E14进行了表达和纯化。经ELISA鉴定，重组单域抗体E14可与EGFRvIII胞外区蛋白产生抗原抗体结合反应，具有较高的亲和力。说明制备的EGFRvIII特异性噬菌体单域抗体库具有较高的库容和多样性，且筛选得到的单域抗体具有较高的抗原活性和免疫学反应性，为今后以EGFRvIII为靶点的恶性肿瘤的诊断和治疗提供新的实验依据。

关键词:EGFRvIII;EGFRvIII胞外区蛋白;重组腺病毒;单域抗体;抗体库

Abstract:

The aim of this study was to construct a recombinant adenovirus expressing extracellular domain gene of human epidermal growth factor receptor variant Ⅲ (EGFRvIII ECD), and to prepare single domain antibody targeting EGFRvIII ECD by immunizing camels and constructing phage display antibody library. Total RNA was extracted from human prostate cancer cell line PC-3 cells and reversely transcribed into cDNA. EGFRvIII ECD gene was amplified using cDNA as template, and ligated into pAdTrack-CMV plasmid vector and transformed into E. coli BJ5183 competent cells containing pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus expressing EGFRvIII ECD was obtained through transfecting the plasmid into HEK293A cells. The recombinant adenovirus was used to immunize Bactrian camel to construct EGFRvIII ECD specific single domain antibody library. The single domain antibody was obtained by screening the library with EGFRvIII protein and the antibody was expressed, purified and identified. The results showed that recombinant adenovirus expressing EGFRvIII ECD was obtained. The capacity of EGFRvIII specific phage single domain antibody library was 1.4×10⁹. After three rounds of enrichment and screening, thirty-one positive clones binding to EGFRvIII ECD were obtained by phage-ELISA, and the recombinant single domain antibody E14 with highest OD₄₅₀ value was expressed and purified. The recombinant E14 antibody can react with EGFRvIII ECD with high affinity in ELISA assessment. The results indicated that the EGFRvIII specific single domain antibody library with high capacity and diversity was constructed and the single domain antibody with binding activity to EGFRvIII was obtained by screening the library. This study may facilitate the diagnosis and treatment of EGFRvIII targeted malignant tumors in the future.

Key words:EGFRvIII;EGFRvIII extracellular domain;recombinant adenovirus;single domain antibody;antibody library