胶质瘤患者人源性噬菌体抗体基因库的构建

Construction of phage library of the human antibody gene from patients with glioma

目的 构建胶质瘤患者人源性噬菌体抗体基因库。方法 从脑胶质瘤患者外周血淋巴细胞（PBL）中提取细胞总RNA,经逆转录后用套式多聚酶链反应（PCR）分别扩增抗体轻链VK和重链VH基因。先构建VK基因库,并使linker与VK基因连接,再将VH基因克隆人VK基因库,即构建成功单链抗体（ScFv）基因库。随机挑取菌落鉴定后测序。结果 从PBL中扩增出人源性VK和VH基因,并构建了库容量约为2×106的ScFv噬菌体抗体基因库。随机挑取克隆的测序结果表明,测定的VH基因与Kabat的抗体基因库中人抗体VH基因有较高的同源性,证明所克隆的基因属于人抗体可变区基因。结论 本实验构建成胶质瘤患者人源性噬菌体抗体基因库,为进一步筛选入源性抗脑胶质瘤抗体奠定了基础。

Objective To construct human antibody gene library with phage display technology. Methods Total RNA was extracted from peripheral blood lymphocytes ( PBL) of patients with glioma and subjected to reverse transcription PCR. Variable region genes( Vk and VH gene) were amplified from the cDNA by using nested polymerase chain reaction. The VK gene library was first constructed and attached to the linker which could specially connect VK gene and VH gene. The single chain Fv (ScFv) library was built when the VH gene was cloned into VK gene library. Positive colonies were selected randomly and sequenced. Results The human VKand VH gene from PBL of patients with glioma were successfully amplified. The ScFv phage antibody gene library in about 2 ×106 capacity was successfully constructed. The sequencing results indicated that the cloned genes encoded variable regions of heavy chains of the human antibody gene and were highly homologous with human heavy chain group of Kabat's database. Conclusion The human antibody gene library is successfully constructed with phage display technology. The present study can be considered as a foundation for the next screening of specific antibody against human gliomas.