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本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础。我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性。结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性。上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础。
The purpose of this study was to construct a non-immune heavy chain single-domain antibody library of alpaca, to identify the diversity of antibody library, and lay the foundation for further screening of antigen-specific heavy chain antibody. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of immunized alpacas. After RNA extraction, specific fragments of VHH were amplified by RT-PCR. The two-step method was used to connect the heavy chain antibody variable region fragment with the phagemid vector pCANTAB5E to obtain a recombinant, and the VHH antibody gene library was obtained after multiple electrotransformation of the competent E. coli TG1. The antibody library capacity was determined by dilution counting and randomized Pick the clone sequencing to verify the diversity of antibody library. The results showed that the library size of the non-immune heavy chain single domain antibody library of alpaca was 1.5 × 109, and the random amplified polymorphic DNA (PCR) showed good diversity. The percentage of independent clones was 80% High homology. The above results show that we have successfully constructed a large volume of alpaca non-immune heavy chain single domain antibody library, and lay the foundation for further screening of antigen-specific heavy chain antibodies.