论文部分内容阅读
[目的]获得骆驼来源的与人CD133抗原特异性结合的纳米抗体。[方法]从3峰新疆双峰驼外周血中分离淋巴细胞,采用巢式PCR扩增获得cDNA并构建VHH天然噬菌体展示文库。包被CD133-606于ELISA板,对天然文库进行3轮亲和筛选,菌落PCR和基因测序确定能结合CD133的候选克隆。挑选重复序列VHH克隆构建到pET28a并转BL21用IPTG诱导表达。IMAC方法纯化重组VHH抗体蛋白,Western Blot和ELISA检测与CD133的抗原结合性。[结果]从109淋巴细胞中构建了库容为1.4×109cfu的噬菌体展示文库,Western Blot及ELISA结果显示重复4次核苷酸序列的VHH-13的纳米抗体能与CD133-606有较好的结合活性。[结论]通过对VHH天然展示文库的亲和筛选及鉴定,成功获得了能与人重组CD133胞外区特异结合的纳米抗体。
[Objective] The aim was to obtain camelid-specific Nanobodies that specifically bind to human CD133 antigen. [Method] Lymphocytes were isolated from the peripheral blood of Bactrian camel 3 peaked in Xinjiang. The cDNA was amplified by nested PCR and the VHH natural phage display library was constructed. CD133-606 was coated on ELISA plates, 3 rounds of affinity screening of natural libraries, colony PCR and gene sequencing to identify candidate clones that could bind to CD133. The repeat VHH clones were selected and constructed into pET28a and transformed into BL21 to induce expression with IPTG. IMAC method to purify recombinant VHH antibody protein, Western Blot and ELISA detection of CD133 antigen binding. [Results] A phage display library with a capacity of 1.4 × 109 cfu was constructed from 109 lymphocytes. The results of Western Blot and ELISA showed that the VHH-13 Nanobody with the nucleotide sequence repeated 4 times could bind well to CD133-606 active. [Conclusion] Nanobodies that specifically bind to the extracellular domain of human recombinant CD133 were successfully obtained by affinity screening and identification of VHH natural display library.