【摘要】 目的克隆和表达人的神经元特异性烯醇化酶(HuNSE)基因,制备HuNSE多克隆抗体,以期用于朊病毒病及相关疾病的临床诊断。方法经RT-PCR扩增HuNSE基因和测序验证后,将其克隆于原核表达载体pQE30,在大肠杆菌M15中诱导表达HuNSE蛋白,蛋白经Ni-NTA亲和纯化后,免疫兔子,所制备的抗血清用ELISA、Westernblotting和免疫组化鉴定。结果所表达的HuNSE蛋白相对分子质量约为22000,以其为抗原制备的HuNSE特异性抗血清具有良好的免疫反应性。Westernblotting和免疫组化结果显示抗血清可识别重组和不同哺乳动物脑组织中的NSE蛋白。结论NSE基因在大肠杆菌中获得了高效表达,所制备的NSE特异性抗血清可用于朊病毒病及其他神经退行性疾病的诊断和研究。更多还原

【Abstract】 Objective To clone and express human neuron-specific enolase(HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.Methods HuNSE gene was amplified by RT-PCR and subcloned into a HIS-tagged expression vector pQE30 after sequence verification.HIS-NSE fusion protein expression was obtained in E.coli M15 after IPTG induction followed by purification of the fusion protein by Ni-NTA affinity chromatography.Two male rabbits were immunized for 4 times with the purified protein,and the antiserum against NSE protein was collected and evaluated by enzyme-linked immunosorbent assay(ELISA),Western blotting and immunohischemistry.Results SDS-PAGE assay yielded an approximately 22 kD HIS-NSE fusion protein.The prepared antiserum could recognize both recombinant NSE protein and native NSE protein extracted from the brain tissues of different mammalian species as shown by Western blotting and immunohischemistry.Conclusion High expression of HuNSE is obtained in E.coli and the prepared antiserum against HuNSE can be used potentially for diagnosis of prion-associated diseases and other nervous degeneration diseases.更多还原