人CD59基因克隆、原核表达及其多克隆抗体的制备

Clone,prokaryotic expression and pre-paration of polyclonal antibody of hCD59

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。

AIM： To construct full length hCD59 eukaryotic and extracellular domain of hCD59（hsCDS9） prokaryotic expression vectors and prepare polyclonal antibody of hCD59. METHODS： cDNA fragments encoding hCD59 and hsCD59 were amplified from human PBMCs by RT-PCR and cloned into the eukaryotic vector pVAX-1 and prokaryotic vector pGEX-KG, respectively. The recombinant fusion protein GST-hsCD59 was expressed in E. coil BL21 induced by IPTG. Then the fusion protein was purified and identified. Polyclonal antibody against hCD59 was prepared by immuni- zing rabbit with pVAX-1-hCD59 and boosting with GST- hsCD59 fusion protein, and the titer was identified. RE- SULTS： The recombinant eukaryotic vector pVAX-1-hCD59 and prokaryotic vector pGEX-KG-hsCD59 were successfully constructed. The GST-hsCD59 fusion protein was over-expressed in E. coli BL21 and the relative molecular mass （Mτ） of the expression product was identical with predicted size. The titer of the anti-hCD59 serum was 1：3 2,00. CONCLUSION： We got the recombinant eukaryotic vector pVAX-1-hCD59, prokaryotic vector pGEX-KG-hsCD59 and rabbit anti-hCD59 polyclonal antibody successfully. These work would be helpful for the further study of the biological function of human CD59.