人源性阿尔茨海默病噬菌体单链抗体库的构建

摘要

Objective To construct the human Alzheimer's disease (AD) specific single chain anti‐bodies (scFv) library for sceening human AD scFv to Aβ1‐42 oligomers .Methods RNA was isola‐ted from 40 ml peripheral blood taken from 18 AD patients .Variable heavy (V H ) and variable light (VL ) genes were amplified by RT‐PCR and linked to the scFv fragments that were then cloned into the phage vector pCANTAB5E .The scFv library was constructed by electroporating E .coli TG1 cells into the pCANTAB5E and rescuing the assistant phage M13K07 .Results The total RNA was extracted .The VH and VL genes were amplified .Electropharesis showed that the length of VH and VL genes was 360bp and 300bp respectively and the length of linked scFv was 750bp .The 2 .4 × 109 scFv library was thus constructed and identified by BstN I digestion .Elec‐tropharesis showed that the length of BstN I‐digested scFv fragments varied ,indicating that the library is of a good diversity .Conclusion The human AD phage scFv library we constructed lays a foundation for screening the antibodies to Aβ1‐42 oligomers and the treatment of AD .%目的：构建人源性阿尔茨海默病（AD）噬菌体单链抗体（scFv）库，为筛选β淀粉样蛋白（Aβ1‐42）的人源性特异性抗体奠定基础。方法采集18例AD患者的外周血40 ml ，提取总RNA ，应用RT‐PCR法得到人抗体可变区重链（V H ）和可变区轻链（V L ）基因。将 V H 和 V L 由连接肽连接得到 scFv 片段，将所得片段双酶切后，克隆至pCANTAB5E噬菌体载体，大肠埃希菌TG1感受态细胞经电击转化，辅助噬菌体 M13K07拯救后构建scFv型噬菌体抗体库。结果总RNA经逆转录PCR扩增VH和VL可变区基因的凝胶电泳显示，PCR产物长度分别为360 bp和300 bp ，其连接形成的scFv片段长度为750 bp ，最终构建了库容为2．4×109的scFv库。BstNⅠ酶切鉴定构建的scFv库，经凝胶电泳可见，酶切片段长度差异性大，显示scFv库具有良好的多样性。结论成功构建人源性AD噬菌体scFv库，为进一步筛选Aβ特异性抗体，继而为AD的治疗研究奠定基础。