抗华支睾吸虫噬菌体抗体库的构建

CONSTRUCTION OF A PHAGE DISPLAY LIBRARY OF Fab ANTIBODIES AGAINST CLONORCHIS SINENSIS

目的 构建抗华支睾吸虫噬菌体抗体库 ,以期筛选出抗循环抗原的抗体组合。方法 应用噬菌体表面呈现技术 ,从感染华支睾吸虫病人淋巴细胞中提取总 RNA,逆转录成 c DNA后 ,用相应引物 PCR扩增出重链 Fd和轻链基因 ,经 Xho 和 Spel、Sac 和 Xba 双酶切 ,先后克隆入噬粒载体 p Comb3,再电转化大肠杆菌 XL1- blue菌株 ,辅助噬菌体 VCSM13超感染。结果 从感染华支睾吸虫病人淋巴细胞中扩增出约 70 0 bp重链 γ1 、γ3Fd段和轻链 κ、λ基因 ,分别和 p Com b3连接后成功导入 XL 1- blue,得到滴度为 7.5× 10 1 0 cfu/ ml,库容量为 5 .6× 10 6 噬菌体抗体库。结论 抗华支睾吸虫 Fab段噬菌体抗体库的构建 ,为进一步筛选抗华支睾吸虫循环抗原单克隆抗体奠定了基础。

The total RNA extracted from PBLs of 16 patients infected with Clonorchis sinensis was reverse transcripted,and Immunoglobulin heavy chain Fd genes and light chain genes were amplified by PCR using general primers scanning γ 1、γ 3 Fd and κ、λ chain.After digestion with XhoI+SpeI and SacI+XbaI,the amplified Fd and light chain fragments were cloned into phagemid pComb3 and electrotransinfected competent E.coli XL1-blue respectively.The phage antibodies were displayed on the surface of recombinant phage after rescue by helper phage VCSM13.By then,a 7.5×10 10clone phage Fab library against Clonorchis sinensis was constructed by using phage surface display technique.