天然的人源性核糖体展示抗体库的构建与初步鉴定

Construction and Verification of a High-capacity Human Single-chain Fv Antibody Library by Ribosome Display

目的构建库容量大、多样性好的核糖体展示单链抗体(ScFv)库,为进一步筛选ScFv奠定基础。方法从健康自愿者的外周血淋巴细胞提取mRNA,经RT-PCR分别扩增抗体重链可变区(VH)和轻链可变区(VL)DNA,进而连接形成ScFvDNA。将其连接T-Vector转化E.coli JM109大肠埃希菌,经蓝白筛选,挑取9个阳性克隆测序以鉴定ScFv组装。通过体外转录和翻译对抗体文库进行初步鉴定。结果VH、VL和ScFvDNA分别约为350、650和1100bp。本试验成功构建ScFv核糖体展示模板。结论成功构建天然的大容量核糖体展示人源性ScFv文库,为下一步利用亲和富集筛选技术获得各类ScFv奠定基础。

Objective To construct a human-derived ribosome display single-chain fragment variant （SeFv） library with great capacity and diversity for further selection of ScFv antibody with high affini- ty. Methods mRNA was isolated from peripheral blood lymphocytes of healthy volunteers. VH and VL DNAs of the antibody were amplified respectively and assembled into ScFv DNAs with a linker DNA by PCR. Then, the ScFv DNAs were ligated to T-vector and transformed into E. coli JM 109. By bluewhite screening, 9 positive clones were selected and sequenced to identify SeFv＇s assembly. Finally, with in vivo transcription/translation, the ScFv antibody of LPS was initially identified. Results DNAs of VH, VL and ScFv were observed to be 350, 650 and 1 100 bp respectively. The ScFv ribosome display model was successfully constructed. Conclusion This library with great capacity could be used to prepare for further selection of ScFv of great varieties.