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目的获取重组人CL-L1颈区-糖识别区纯化蛋白,制备多克隆抗体。方法采用PCR方法扩增人CL-L1颈区-糖识别区目的基因片段,构建原核表达载体,进行原核表达与蛋白纯化,然后免疫家兔,制备多克隆抗体。采用ELISA检测抗体效价,免疫印迹法检测蛋白纯度和抗体的特异性。结果成功构建了人CL-L1颈区-糖识别区原核表达载体pRSET-C/NECK-CRD△K,在E.coliBL21中表达,镍亲和层析柱纯化,得到相对分子质量19 200的融合蛋白,免疫家兔后收获抗血清,ELISA显示抗体效价为1/20 000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组人CL-L1颈区-糖识别区蛋白特异性结合。结论本研究获得了人CL-L1颈区-糖识别区纯化蛋白,制备了人CL-L1颈区-糖识别区多克隆抗体,为CL-L1的结构、组织表达谱和功能的研究奠定了基础。
Objective To obtain the purified protein of CL-L1 cervical-saccharide recognition region of recombinant human and prepare polyclonal antibody. Methods The gene fragment of human CL-L1 cervical-sugar recognition region was amplified by PCR and the prokaryotic expression vector was constructed. The prokaryotic expression vector and protein were purified, and then the rabbits were immunized to prepare polyclonal antibody. Antibody titers were detected by ELISA and protein purity and antibody specificity were determined by Western blotting. Results The prokaryotic expression vector pRSET-C / NECK-CRD △ K of CL-L1 cervical-sugar recognition region was successfully constructed and expressed in E.coli BL21. The fusion protein was purified by nickel affinity chromatography to obtain a fusion protein with a relative molecular mass of 19 200 The antiserum was harvested after immunized rabbits. ELISA showed that the antibody titer was 1/20 000, which was highly specific. The results of immunoblotting showed that the prepared polyclonal antibody was specific for the protein of the human CL-L1 cervical-sugar recognition region Combined. Conclusion In this study, we obtained the human CL-L1 cervical-glycoprotein purified protein and prepared the human CL-L1 cervical-glycocated polyclonal antibody, which laid the foundation for the study of CL-L1 structure, tissue expression profile and function basis.