抗人B-淋巴细胞刺激因子噬菌体单链抗体库的构建

目的构建抗人B-淋巴细胞刺激因子噬菌体单链抗体库。方法从B-淋巴细胞刺激因子免疫小鼠的脾细胞中抽提总RNA,经逆转录聚合酶反应合成cDNA,分别扩增小鼠重链可变区基因和轻链可变区基因。通过重叠延伸拼接法将重链可变区基因和轻链可变区基因组装成单链抗体基因,重组于载体pFUSE5,电转化E.coliXL 1-Blue。在辅助噬菌体援救下,构建成噬菌体单链抗体库。采用聚合酶链反应、核苷酸序列测定和酶联免疫分析鉴定单链抗体库的特征。结果本抗体库的噬菌体库容量约1×106,单链抗体基因插入效率为93%,存在序列差异性并含有抗B-淋巴细胞刺激因子单链抗体。结论成功地构建了抗人B-淋巴细胞刺激因子单链噬菌体抗体库,为筛选抗B-淋巴细胞刺激因子单链抗体药物奠定了良好的基础。

Construction of a scFv phage display library from a mouse immunized with human B lymphocyte stimulator

Objective To construct a phage displayed single-chain antibodies(scFv) library against BLyS by phage display technology.Methods Total RNA extracted from the spleen cells of immunized BALB/c mouse with BLyS was purified and reverse transcriptase-polymerase chain reaction(RT-PCR) was used to amplify the heavy chain variable regions(VH) and light-chain variable regions(VL) genes respectively,which were subsequently assembled into scFv gene with a linker through SOE(splicing by overlap extension) ligation.The scFv genes were cloned into vector pFUSE5 and electroporated into Escherichia coli XL1-Blue.Rescued by VCSM13 helper phage,the scFv phage display library was generated.A polymerase chain reaction(PCR),a nucleotides sequence analysis,and an enzymelinked immunosorbent assay(ELISA) were performed to evaluate the constructed scFv library.Results The constructed phage display scFv library had a capacity of approximately 1×106,with 93% of the recombinant vectors containing scFv gene insertion.DNA different sequences came from random clones showed the diversity of the library.ELISA results indicated the library contain scFvs binding to BLyS antigen.Conclusions A phage display scFv library against BLyS is successfully constructed.It establishes a better base for the development of antibody drug for therapy.