论文部分内容阅读
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。
Objective To clone and express human neuron specific enolase (HuNSE) gene and prepare HuNSE polyclonal antibody for the clinical diagnosis of prion diseases and related diseases. Methods After HuNSE gene was amplified by RT-PCR and verified by sequencing, it was cloned into prokaryotic expression vector pQE30 and expressed in Escherichia coli M15. HuNSE protein was purified by Ni-NTA affinity purification, and then the rabbit was immunized. Serum was identified by ELISA, Western blotting and immunohistochemistry. As a result, the expressed HuNSE protein had a relative molecular mass of about 22000, and HuNSE-specific antiserum prepared from the antigen showed good immunoreactivity. Western blotting and immunohistochemistry showed that the antisera recognized NSE protein in recombinant and different mammalian brain tissues. Conclusion NSE gene was highly expressed in Escherichia coli. The NSE-specific antiserum could be used in the diagnosis and research of prion diseases and other neurodegenerative diseases.