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目的构建人前蛋白转化酶枯草溶菌素9(pcsk9)的原核表达载体并诱导其表达,制备其蛋白的多克隆抗体。方法聚合酶链式反应扩增人工合成的pcsk9 cDNA序列;扩增产物经TA克隆、双酶切、连接,构建pET-28b-pcsk9表达载体;将重组质粒转化入大肠杆菌表达菌BL21(DE3)的感受态细胞中,诱导表达;用纯化检测过的目的蛋白免疫新西兰兔,最后采集全血收集血清,获得多克隆抗体,并对该抗体进行免疫印迹及效价测定。结果 PET-28b-pcsk9原核表达载体构建成功,用诱导表达的目的蛋白免疫动物,收集血清获得多克隆抗体。对多克隆抗体进行纯化,免疫印迹检测结果证明自制多克隆抗体特异性良好,效价测定为1:13 200。结论成功构建了pcsk9原核表达载体pET-28b-pcsk9,且获得了其蛋白的多克隆抗体。
Objective To construct a prokaryotic expression vector of human pre-protein convertase subtilisin 9 (pcsk9) and induce its expression to prepare its polyclonal antibody. Methods The pcsk9 cDNA sequence was amplified by polymerase chain reaction (PCR). The amplified product was cloned by TA, double digested and ligated to construct pET-28b-pcsk9 expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) Of the competent cells, induced expression; purified purified target protein New Zealand rabbit, and finally collected whole blood collected serum to obtain polyclonal antibodies, and the antibodies were immunoblotting and titer determination. Results The prokaryotic expression vector PET-28b-pcsk9 was successfully constructed. The target protein was induced to immunize animals and the serum was collected to obtain polyclonal antibodies. The polyclonal antibody was purified and the results of immunoblotting showed that the homemade polyclonal antibody was of good specificity and the titer was 1:13 200. Conclusion The pcsk9 prokaryotic expression vector pET-28b-pcsk9 was successfully constructed and its polyclonal antibody was obtained.