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.

    Construction of ouabain single┐chain Fv phage anti┐body library by phage display

    YUAN Wei-Qing WANG Hao LU Zhuo-Ren CHEN Ping HAN Hua NIU Li-Guo

    1 Department of Cardiology，First Hospital of Xi'an Jiaotong University，Xi'an710061，China，

    2 Department of Biochemistry，Fourth Military Med-ical University，Xi'an710033，China

    Keywords:ouabain;phage display;antibodies

    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 ......