关键词: 嗜菌体抗体库;单链抗体;重组

    中图号:Q782

    摘 要:目的 构建一个大库容量(>108 )的人类天然单链抗体库. 方法 从正常人400mL外周血分离淋巴细胞，提取mRNA后反转录出cDNA第一链，行半套式PCR扩增VH 和VL 基因片段，依次插入含Loxp和Loxp511序列的pDAN5，电转化TG1大肠杆菌，构建初级库进一步用初级库感染BS1365使其VH ，VL 发生重组从而获得次级库. 结果 所有VH ，VL 亚类基因都得到了扩增，scFv克隆效率为99.9%，重组效率为每个单克隆BS1365中含至少8种不同的scFv基因.初级库容量为107 ，次级库容量至少为1011 . 结论 我们采用细胞内重组的方法构建了1011 的人类天然抗体库.

    Construction of a large human naive phage library

    GUO Qing-Dong，ZHANG Xiang，FEI Zhou，ZHAO Ai-Zhi，WANG Yan-Gang，WU Jing-Wen，LI Xia，ZHANG Yong-Qin

    Institute of Neurosurgery of Chinese PLA，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China

    Keywords:phage antibody library;scFv;recombination

    Abstract:AIM To construct a large human naive phage dis-play library.METHODS Lymphocytes were isolated from400mL blood，which was obtained from two healthy donor.mRNA was isolated and VH ，VL fragments were amplified by semi-nest PCR，which were individually cloned into the phagemid pDAN5containing the Loxp and Loxp511se-quences.The primary library was constructed by electropo-rating TG1with the phagemid containing scFv fragments.BS1365were infected by the primary library and the second library was obtained.RESULTS All the fragments of the subgroup of the VH and VL families were amplified.The cloned efficiency of scFv was99.9% ......