基于RT-PCR基础上的体内重组法构建人源性胶质瘤单链抗体库

Construction of phage library of human glioma single chain fragment variable region antibody by in vivo recombinaton based on RT-PCR

目的 构建人源性胶质瘤噬菌体抗体库 .方法 经逆转录、套式PCR从脑胶质瘤患者血淋巴细胞中扩增出抗体轻链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 ,即构建成约为 3.5× 10 6 的单链抗体 (ScFv)基因库 .以Loxp5 11序列替代Linker序列 ,并将ScFv克隆入pDNA5内进行重组、鉴定 ,得到新的ScFv噬菌体抗体库 .结果 表明原初级噬菌体抗体库经Cre Loxp5 11系统重组后库容量达到 8.5× 10 8,部分测序证实抗体基因与人源性抗体基因数据库同源 .结论 利用体内重组系统明显提高了所建的抗体库容量 。

AIM To construct large library of human glioma antibody by phage display technology. METHODS Total RNA was extracted from peripheral blood lymphocytes of patients with glioma and reversely transcripted into cDNA. Variable region genes (V κ and V H gene) were amplified from the cDNA using nested polymerase chain reaction. V κ gene library was firstly constructed and attached to the Linker which can specially connect V κ gene with V H gene, then V H gene was cloned into V κ gene library to build ScFv library. Positive colonies were randomly selected and analyzed by sequencing. Results implied that amplified human V κ gene and V H gene were from PBL of patients with glioma. ScFv phage antibody library capacity was about 3.5×10 6. Then the linker was replaced by Loxp511 ,and new ScFv genes were cloned into pDNA5 by in vivo recombination. RESULTS The primary library was successfully enlarged .The capacity of new library was 8.5×10 8.The results of 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 I of Kabats database. CONCLUSION The Cre Loxp511 vivo recombination system could enlarged small primary library effectively. The present study may be used as a foundation for next screening specific antibody against human glioma.