大库容量人源性天然单链抗体库的构建

目的：构建一个大库容量（＞10^8）的人类天然单链抗体库。方法：从正常人400mL外周血分离林巴细胞，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pDNA5，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10^7，次级库容量至少为10^11。结论：我们采用细胞内重组的方法构建了10^11的人类天然抗体库。

Construction of a large human naive phage library

AIM To construct a large human naive phage display library. METHODS Lymphocytes were isolated from 400 mL blood, which was obtained from two healthy donor. mRNA was isolated and V H, V L fragments were amplified by semi nest PCR, which were individually cloned into the phagemid pDAN5 containing the Loxp and Loxp511 sequences. The primary library was constructed by electroporating TG1 with the phagemid containing scFv fragments.BS1365 were 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 was 99.9%, recombination in vivo was 8 scFv per BS1365 clone. The primary library was 10 7 and the second library was up to 10 11 . CONCLUSION A large human naive library of 10 11 could be obtained by the technique of recombination in vivo .