单域抗体T7噬菌体展示文库构建与鉴定

Construction and identification of nanobody T7 phage display library

优化噬菌体展示文库构建策略,获得高品质羊驼源天然纳米抗体T7噬菌体展示文库,并对文库质量进行鉴定。分离羊驼外周血淋巴细胞,提取总RNA,RT-PCR扩增羊驼重链抗体可变区(VHH)基因,插入T7 select 415-1b噬菌体载体构建文库,计算文库的库容量,测定文库传代稳定性。测序分析VHH基因的多样性和纳米抗体氨基酸序列特征。Western-blot检测纳米抗体在噬菌体表面的表达情况,电镜观察文库中重组噬菌体颗粒形态。结果表明,构建羊驼源纳米抗体T7噬菌体展示文库,有效库容达到2.73×109 PFU;纳米抗体与羊驼源抗体同源性高,氨基酸序列显示纳米抗体特征区域,并其在噬菌体表面高拷贝表达;重组噬菌体颗粒结构完整,但随着传代出现插入基因的丢失。

To construct a high-quality natural alpaca nanobody phage display library by strategy optimization,and systematically identified the quality of the library,the total RNA was isolated form peripheral blood mononuclear cell of 6 alpacas and then reverse transcripted into cDNA.The VHH gene was amplified and insert into plasmid and T7 select 415-1b vector,respectively,to construct plasmid and phage display library.The phage library was identified by volume calculating,propagating stability testing and sequencing.The expression of nanobody was tested by Western-blot,and the structure of phage particles were detected by electron microscopy.Results showed that the effective volume of phage library was 2.73×109 PFU,and VHH nanobody was successfully displayed on T7 phage surface in high copy number.Sequence analysis showed that VHH nanobody had high homology with alpaca antibody.Recombinant phage presented intact particle structure the same as the donate T7 select 415-1b phage.However,during continuous passage a part of recombinant phages would lost the insert VHH gene,which indicated that only lower-generation phage library could be used for affinity screening.