【摘要】 目的　应用噬菌体展示技术构建钠泵抑制物单链抗体库 .方法　用 OUA- OVA(ouabain- ovalbumin)免疫小鼠 ,取脾脏 ,分离淋巴细胞 ,提取 m RNA,逆转录成 c DNA第一链 ,用简并引物经 PCR分别扩增轻、重链抗体库 ,经重叠延伸PCR由 L inker将轻、重链拼接成 Sc Fv(单链抗体 ) ,用 RS引物以 Sc Fv为模板进行第 2次 PCR,同时在 5 及 3 端分别加上 Sfi I,N ot I酶切位点 .经 Sfi I,N ot I酶切后 ,在 T4连接酶的作用下将 Sc Fv与 p CANTAB 5 E噬菌粒连接 ,电转化进入TG1大肠杆菌 ,挑取 12个单菌落提取质粒用 Eco R 和 H ind 进行酶切鉴定 ,后经 M13KO7辅助噬菌体拯救 ,离心收集上清即为全套 Sc Fv基因的噬菌体表面展示文库 .结果　以OU A- OVA免疫小鼠 ,检测血清中抗体效价可达 (1∶ 2×10 6 ) ,用通用引物分别扩增出轻、重链片段的大小为 32 5 bp和 34 0 bp,将两者拼接得到约 72 0 bp大小的单链抗体库 ,以8× 10 1 1 的转化效率转染 TG1细胞 ,Eco R 和 H ind 酶切后 ,12个单菌落所提噬菌粒 DNA中有 10个切出了约为 2 .1kb的目的条带 ,所构建的噬菌体抗体库库容量约为 1.2× 10 8.经 M13KO7超感染后 ,测噬斑滴度为 9× 10 1 4 pfu· L- 1 .结论　成功地构建了钠泵抑制物噬菌体单链抗体库 .更多还原

【Abstract】 AIM To construct a ouabain ScFv(single chain fragment variable) antibody library by phage display. METHODS Mice were immunized by OUA OVA(ouabain ovalbumin). mRNA was isolated from the spleen lymphocyte and reversely transcripted. The heavy chain and kappa light chain variable region genes (V H and V L) repertoirs of immunoglobin were amplified individually and assembled into ScFv by a linker with overlap extension PCR. ScFv was reamplified with RS primer, meantime S fi I and NotI restriction sites were added to the ScFv different ends. ScFv was digested by S fi I and NotI, and cloned into the phagemid pCANTAB 5E and introduced into E.coli TG1 by electroporation. Phagemid containing bacterial colonies were infected with M13KO7 and the ScFv fusion protein was displayed on the surface of recombinant phage. RESULTS Titer of rat serum was 1.2×10 6 after being immunized with OUA OVA. The V H, V L fragment was 340 bp and 325 bp, respectively. After assembly, the ScFv was about 720 bp. The library capacity was 1.2×10 8. Ten clones of twelve clones contained 2.1 kb fragment after EcoRI and Hin dⅢ digestion. Transfection test indicated that the titer of the culture supernatant was about 9×10 14 pfu·L -1 . CONCLUSION A phage display library of ouabain ScFv antibody has been constructed successfully.更多还原