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目的针对CD26酶催化结构域制备多克隆抗体。方法应用RT-PCR技术以人白细胞mRNA为模板,扩增获取编码CD26催化结构域的基因序列,克隆入原核表达载体PET32a后,转化BL21感受态细菌,经IPTG诱导表达得到his-CD26融合蛋白;亲和层析柱纯化并经Western-blot鉴定后,用此重组蛋白免疫2只新西兰纯种大白兔,获取免疫血清共180 ml,经蛋白A柱纯化及抗原抗体亲和纯化后,采用间接ELISA法检测抗体效价,Western-blot及免疫细胞化学染色进行抗体效价及特异性鉴定。结果构建出PET32a/CD26原核表达质粒,并在大肠杆菌BL21中获得高效表达,经his亲合层析纯化后蛋白量达2.3 mg/ml;制备的多抗血清纯化后效价达256 000,经Western-blot证明能特异性的识别CD26重组蛋白,免疫细胞化学染色显示能特异的结合于H9细胞。结论成功制备了能特异性识别CD26的多克隆抗体。
Objective To prepare polyclonal antibodies against the CD26 enzymatic domain. Methods Human leukocyte mRNA was used as a template to amplify the cDNA sequence encoding CD26 catalytic domain by RT-PCR. After cloned into prokaryotic expression vector PET32a, the competent cells of BL21 were transformed and induced by IPTG to obtain his-CD26 fusion protein. After purification by affinity chromatography and identification by Western-blot, two New Zealand purebred rabbits were immunized with the recombinant protein, and 180 mL of immune serum was obtained. After purified by protein A column and affinity-purified by antigen-antibody, indirect ELISA Antibody titers, Western-blot and immunocytochemistry were used to detect antibody titer and specificity. Results The prokaryotic expression plasmid pET32a / CD26 was constructed and expressed in E. coli BL21. After purification by his affinity chromatography, the protein level reached 2.3 mg / ml. The titer of purified multi-antiserum was 256 000, Western-blot showed that the recombinant protein could specifically recognize CD26, and immunocytochemical staining showed that it could specifically bind to H9 cells. Conclusion Polyclonal antibodies that specifically recognize CD26 were successfully prepared.