驼源天然单域重链抗体库的构建与鉴定

从未经主动免疫的健康羊驼(Lama pacos)外周血淋巴细胞中提取总RNA,反转录后作为第一轮PCR的模板。根据重链抗体保守区域设计引物,经巢式PCR法扩增获得了全套重链抗体可变区基因,将其克隆至噬菌粒pHEN1,电转化大肠杆菌TG1得到初级抗体库NAL,含有2×10⁷个独立克隆,菌落PCR和Hinf I酶切分析结果显示,克隆效率大于97%,文库的多样性良好。辅助噬菌体救援后,得到噬菌体展示文库命名为NA-PDL,滴度达10¹³CFU/ml。以真菌毒素人工抗原DON-MBSA为目标抗原,对NA-PDL进行了淘选,第二轮洗脱物中,阳性克隆率达36.4%,提示针对目标抗原的噬菌体颗粒得到了有效富集,文库NA-PDL多样性较好,为后续淘选针对特定抗原的单域重链抗体奠定了基础。

Construction and Biopanning of Camelid Naive Single-domain Antibody Phage Display Library

The objective is to construct a camelid nave single-domain heavy chain antibody phage display library. Total RNA was purified from 30ml blood of two healthy non-immune alpacas (Lama pacos) and directly used for complementary DNA (cDNA) synthesis. Three sets of primers were designed based on the conserved region of heavy-chain antibody. The repertoire of VHH coding sequence was amplified by nested PCR, and the PCR products were cloned into a phagemid vector pHEN1. By electroporation of E.coli TG1, the primary library (designate NAL) was obtained containing more than 10⁷ independence clones. After helper phage rescue, the phage display library (designate SNA-PDL) was generated with a titre up to 10¹³ CFU/ml. The library exhibited high diversity as judged by the Hinf I restriction pattern. Solid phage biopanning against artificial antigen DON-MBSA showed significant enrichment of binding phage particles. The positive rate of panning round two was 36.4%. The data indicated that a nave single-domain antibody phage display library was constructed, which has good diversity and would be useful for generating VHHs with specific binding affinity.