抗己烯雌酚噬菌体单链抗体库的构建

Construction of phage display library of single-chain Fv antibodies against diethylstilbestrol

利用噬菌体表面展示技术、绕过杂交瘤技术,提取经DES-BSA免疫的BALB/c小鼠脾细胞总RNA,用相应引物扩增小鼠免疫球蛋白VH基因和VL基因,构建VH、VL抗体库。通过重叠延伸PCR(SOE-PCR),以(Gly4Ser)3为连接肽,将VH基因和VL基因连接成VH-Linker-VL,在SfiⅠ+NotⅠ酶切位点定向插入pCANTAB-5E噬菌体载体,转化大肠杆菌TG1后,经M13KO7辅助噬菌体拯救,构建成了全套抗己烯雌酚单链抗体表面展示文库。噬菌体中scFv基因的插入率为87.5%,经M13KO7超感染后,其噬斑计数的效价为2.2×1011PFU/mL。结果表明,抗己烯雌酚的单链抗体库的构建,为进一步富集筛选并表达己烯雌酚单链抗体奠定了基础。

To construct single-chain variable fragment（scFv） phage antibody library associated with diethylstilbestrol（DES） ,the total RNA was extracted from spleen cells of mouse with the highest antiserum titers. The variable regions of heavy chain（VH） and light chain（VL） from the spleen cells were connected with a flexible linker--（Gly4 Ser）3 using SOE technique. The purified scFv gene repertoire was di gested with restriction enzymes of Sfi I/Not I . It was cloned into a phagemid pCANTAB-5E, and then transformed into Escherichia coli TG1 cells. The transformed bacteria were rescued by M13KO7 helper phage and the anti-DES phage antibody libraries were successfully constructed. Randomly digestive reactions showed that the positive insert ratio was 87.5%. The titer of plaque was 2.2 × 10^11 PFU/mL after superinfection with M13KOT.