噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}

Construction of Phage Display Antibody Library to MCF-7 Cells and Screening of the Single-chain Antibodies Against Breast Cancer Cells

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.