驼源天然纳米抗体噬菌体展示库的构建及抗CD19纳米抗体的筛选、表达及鉴定

Construction of camelidae natural nanobody phage dispaly library and screening,production and identification of anti-CD19 nanobody

目的构建驼源天然纳米抗体噬菌体展示文库并进行验证,用于筛选针对不同抗原的纳米抗体,并以CD19为抗原筛选靶向纳米抗体,进行表达并验证。方法对提取的双峰驼脾脏总RNA进行反转录并通过巢式PCR获得其重链可变区基因片段,构建至pCANTAB5e噬菌粒载体中,电转化至TG1大肠杆菌构建纳米抗体噬菌体展示库,以辅助噬菌体KM13进行救援后,分析库容及多样性。以CD19为抗原,采用生物素化抗原与链霉亲和素磁珠相结合的方法进行纳米抗体的淘筛,对淘筛后的单克隆进行ELISA鉴定、测序、分析、表达和验证。结果构建的纳米抗体噬菌体展示库有较好的多样性,片段插入率接近100%,随机挑取的20个克隆中其氨基酸同源性为65.85%,经辅助噬菌体救援后展示库滴度为9.0×10^13集落形成单位(CFU)/mL。对以CD19为抗原淘筛后获得的阳性克隆进行测序分析,并对纳米抗体进行表达与验证,结果显示具有与CD19结合的能力。结论成功构建滴度高、多样性好的驼源天然纳米抗体噬菌体展示库,以CD19为抗原进行淘筛获得三条抗CD19纳米抗体序列,为研究以CD19为靶标的诊断试剂盒以及抗体药物提供技术支持,为制备靶向CD19的嵌合抗原受体T(CAR-T)细胞提供基础。

Objective To construct and verify camelidae natural nanobody phage display library for selection of nanobodies against various antigens,and to obtain and identify the nanobody targeting CD19.Methods The total RNA of spleen of Bactrian camel was reverse transcribed and the variable region gene fragment of its heavy chain was obtained by nested PCR.It was constructed into the pCANTAB5e phagemid vector and electrotransformed into TG1 E.coli to develop the natural nanobody phage display library.After rescued by the KM13 helper phage,its capacity and diversity were analyzed and identified.Nanobody against CD19 was screened using biotinylated antigen combined with streptavidin magnetic beads,followed by ELISA,sequencing,exogenous expression and verification.Results The constructed natural phage nanobody display library had great diversity,and its fragment insertion rate was about 100%.The amino acid homology of 20 randomly selected clones was 65.85%,and the titer of the display library rescued by the helper phage was 9.0×10^13 CFU/mL.After panning with CD19 as the antigen,the positive clones were sequenced and analyzed,and finally anti-CD19 nanobody sequences were obtained.The exogenously expressed anti-CD19 nanobody based on the sequences was verified having the ability to bind to CD19.Conclusion A camelidae natural nanobody phage display library with high titer and great diversity has been successfully constructed.Three anti-CD19 nanobody sequences have been obtained by panning with CD19.In addition,this study provides technical support for researching and developing diagnostic kits and antibody drugs targeting CD19,and it is a novel direction to improve CAR-T cells targeting CD19.