摘要
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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第10期924-926,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重大传染病专项基金资助(2008ZX10003-005)