摘要
为构建人源性脑胶质瘤噬菌体抗体库 ,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PCR分别扩增抗体轻链Vκ和重链VH基因。先构建Vκ基因库 ,并使连接Vκ和VH基因的linker与Vκ基因连接 ,再将VH基因克隆入Vκ基因库 ,即构建成功单链抗体 (ScFv)库。随机挑取菌落鉴定 ,测序表明从PBL中扩增出人源性Vκ和VH基因 ,并构建了库容量约为 2× 10 6的ScFv噬菌体抗体库。随机挑取克隆的测序表明 ,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人抗体可变区基因。结果表明实验构建成人源性脑胶质瘤噬菌体抗体库 。
To construct human antibody repertory with phage display technology. Total RNA was extracted from peripheral blood lymphocytes (PBL) of clinical glioma patients and subjected to reverse transcription PCR. Variable region genes(V κ and V H genes) were amplified from the cDNA using nested polymerase chain reaction. V κ gene library was first constructed and attached to the Linker which could specially connect V κ gene and V H gene, then V H gene was cloned into V κ gene library to build ScFv repertoire. Positive colonies were selected randomly and sequenced. The results implied that amplified human V κ gene and V H gene could be derived from PBL of clinical glioma patients. ScFv phage antibody gene repertoire in about 2×10 6 capacity was successfully constructed. The sequencing indicated that the cloned genes encoded variable regions of heavy chains of human antibody and were highly homologous with human kappa heavy chain group III of Kabat's database. Then human antibody repertoire was successfully constructed with phage display technology. The present study can be used as a foundation for succeeding screening of specific antibody against human glioma.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2001年第11期795-797,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助课题 (编号 39970 85 2 )
