Han Dong-gang, Duan Xiao-yi, Guo You-min, Zhou Qi, Wang Quan-ying, Yang Guang-xiao
Department of Medical Ultrasound Study, Second Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710004, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Jan;30(1):25-9.
To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system.
The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody.
The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII.
An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.
通过噬菌体抗体库展示系统获得特异性抗表皮生长因子受体III型变异体（EGFRvIII）单链抗体（ScFv）。
从用pep-3-OVA蛋白免疫的BALB/c小鼠脾脏B细胞中提取总RNA，通过逆转录合成第一链cDNA。扩增抗体VH和VL基因片段，并通过接头连接成ScFv基因。将ScFv基因连接到噬菌粒载体pCANTAB5E中，转化至感受态大肠杆菌TG1。然后用M13KO7辅助噬菌体感染转化细胞以产生重组噬菌体，构建噬菌体ScFv文库。用pep-3-BSA蛋白筛选噬菌体抗体库，并进行ELISA以鉴定抗体活性。
琼脂糖凝胶电泳分析显示抗体的VH和VL基因片段长度分别约为350 bp和320 bp。ScFv基因长度为780 bp，与预期长度一致。拯救出插入ScFv基因的重组噬菌粒，构建了库容为5.0x10(6)的免疫噬菌体ScFv文库。重组ScFv噬菌体滴度为3.0x10(4) cfu/ml，第四次噬菌体收获量是第一次的56倍。SDS-PAGE显示可溶性ScFv的分子量约为28 kD。ELISA结果表明ScFv与EGFRvIII结合具有良好的特异性。
成功构建了免疫噬菌体ScFv文库，ScFv抗体片段能够特异性结合EGFRvIII。
