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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。
Total RNA was extracted from peripheral blood lymphocytes of unhealthy healthy alpaca (Lama pacos) and reverse transcribed to serve as a template for the first round of PCR. Primers were designed according to the conserved region of heavy chain antibody. A complete set of heavy chain antibody variable region genes were amplified by nested PCR. The heavy chain variable region gene was cloned into phagemid pHEN1 and transformed into E.coli TG1 to obtain primary antibody library NAL. 107 independent clones, colony PCR and Hinf I digestion analysis showed that the cloning efficiency was greater than 97%, the diversity of the library is good. After helper phage rescue, the phage display library was named NA-PDL with a titer of 1013 CFU / ml. With mycotoxin DON-MBSA as target antigen, NA-PDL was panned. In the second round of eluate, the positive cloning rate was 36.4%, which indicated that the phage particles targeting the target antigen were effectively enriched. The library The diversity of NA-PDL is good, which lays the foundation for the subsequent panning of single domain heavy chain antibody against a specific antigen.