钠泵抑制物噬菌体单链抗体库的构建_医学论文.docVIP

钠泵抑制物噬菌体单链抗体库的构建_医学论文 钠泵抑制物噬菌体单链抗体库的构建_医学论文 作者：原卫清　王颢　吕卓人　陈萍　韩骅　牛利国 【关键词】 钠泵抑制物 　　关键词: 钠泵抑制物噬菌体展示技术抗体 　　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mRNA，逆转录成cDNA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Linker将轻、重链拼接成ScFv（单链抗体），用RS引物以ScFv为模板进行第2次PCR，同时在5’及3’端分别加上SfiI，NotI酶切位点.经SfiI，NotI酶切后，在T4连接酶的作用下将ScFv与pCANTAB5E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EcoRⅠ和HindⅢ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ScFv基因的噬菌体表面展示文库. 结果 以OUA-OVA免疫小鼠，检测血清中抗体效价可达（1 2×106 ），用通用引物分别扩增出轻、重链片段的大小为325bp和340bp，将两者拼接得到约720bp大小的单链抗体库，以8×1011 的转化效率转染TG1细胞，EcoRⅠ和HindⅢ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1.2×108 .经M13KO7超感染后，测噬斑滴度为9×1014 pfu L-1 . 结论 成功地构建了钠泵抑制物噬菌体单链抗体库. 　　Keywords:ouabainphage displayantibodies 　　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（VH and VL ）repertoirs of im-munoglobin were amplified individually and assembled into ScFv by a linker with overlap extension PCR.ScFv was reamplified with RS primer，meantime SfiI and NotI restric-tion sites were added to the ScFv different ends.ScFv was di-gested by SfiI and NotI，and cloned into the phagemid pCANTAB5E and introduced into E.coli TG1by electropo-ration.Phagemid-containing bacterial colonies were infected with M13KO7and the ScFv fusion protein was displayed on the surface of recombinant phage.RESULTS Titer of rat serum was1.2×106 after being immunized with OUA-OVA.The VH ，VL fragment was340bp and325bp，respectively.After assembly，the ScFv was about720bp.The library ca-pacity was1.2×108 .Ten clones of twelve clones contained2.1kb fragment after EcoRI and HindⅢdigestion.Trans-fection test indicated that the titer of the culture supernatant was about9×1014 pfu L-1 .CONCLUSION A phage display library of ouabain ScFv antibody has been constructed