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目的:构建人CD59真核及原核表达质粒,并制备抗人CD59多克隆抗体,以用于研究糖蛋白CD59生物学功能。方法:用RT-PCR技术从人PBMCs中获取hCD59全长及其胞外区hsCD59cDNA序列,分别克隆至pVAX-1真核表达载体及pGEX-KG原核表达载体。重组融合蛋白GST-hsCD59由经IPTG诱导的大肠杆菌BL21表达后纯化并鉴定。用pVAX-1-hCD59质粒免疫家兔并用纯化的GST-hsCD59融合蛋白加强免疫制备兔抗人CD59多克隆抗体并测定其效价。结果:成功构建人CD59真核表达质粒pVAX-1-hCD59和原核表达质粒pGEX-KG-hsCD59。融合蛋白GST-hsCD59在大肠杆菌中高效表达,表达产物的相对分子质量(Mr)同预期值一致。制备的兔抗人CD59多克隆抗体效价为1∶3200。结论:成功地构建了重组真核表达质粒pVAX-1-hCD59及原核表达质粒pGEX-KG-hsCD59,获得了兔抗人CD59多克隆抗体,这将有助于我们深入研究CD59在人类疾病中的生物学功能。
OBJECTIVE: To construct eukaryotic and prokaryotic expression plasmids of human CD59 and prepare anti-human CD59 polyclonal antibody to study the biological function of glycoprotein CD59. Methods: The hCD59 cDNA sequence of hCD59 and its extracellular domain were obtained from human PBMCs by RT-PCR and cloned into pVAX-1 eukaryotic expression vector and pGEX-KG prokaryotic expression vector respectively. Recombinant fusion protein GST-hsCD59 was purified and identified from IPTG-induced E. coli BL21 expression. Rabbits were immunized with the pVAX-1-hCD59 plasmid and immunized with the purified GST-hsCD59 fusion protein to prepare rabbit anti-human CD59 polyclonal antibodies and their titers were determined. Results: The human CD59 eukaryotic expression plasmid pVAX-1-hCD59 and the prokaryotic expression plasmid pGEX-KG-hsCD59 were successfully constructed. The fusion protein GST-hsCD59 was highly expressed in E. coli, and the relative molecular mass (Mr) of the expressed product was consistent with the expected value. The prepared rabbit anti-human CD59 polyclonal antibody titer was 1:3200. CONCLUSION: The recombinant eukaryotic expression plasmid pVAX-1-hCD59 and the prokaryotic expression plasmid pGEX-KG-hsCD59 have been successfully constructed and the polyclonal rabbit anti-human CD59 antibody has been obtained, which will help us to further study the role of CD59 in human diseases biological functions.