【摘要】 为对SARSN蛋白的功能进行研究,并建立快速、方便的诊断方法,将已克隆、构建成功的重组质粒PGEX-4T/N转化至E.coliBL21中,IPTG诱导表达,表达产物与兔源GST多抗在66kD处有很强的反应性,融合蛋白GST-N分别通过GSTrapFF1mL纯化柱和割胶纯化两种方法获得了具有生物活性和变性的融合蛋白.其蛋白浓度分别为0.85mg/mL和0.433mg/mL.利用纯化的GST-N蛋白免疫SPFBALB/c小鼠,获得4株能稳定传代并分泌抗SARSN单克隆抗体(mAb)的杂交瘤细胞,分别命名为3E5、6B9、4C2、4B7,经Westernblot分析表明,所获得的4株抗SARSN单克隆抗体与纯化的N蛋白均具有特异反应性,且不与禽冠状病毒的N蛋白发生反应.更多还原

【Abstract】 To efficiently express SARS coronavirus （SARS-CoV） nucleocapsid （N） protein in vitro and produce monoclonal antibody （mAb） against SARS-CoV N protein, the recombinant clone PGEX-4T/N was induced with IPTG to express target protein and the fusion protein （GST-N） reacted specifically with anti-GST polyclonal antibody. The expressed recombinant protein was purified by affinity chromatography and gel extraction, where the purified protein concentrations were 0.85 mg/mL and 0.433 mg/mL respectively. Four hybridoma cell lines secreting monoclonal antibody （mAb） against SARS-CoV N protein were obtained and named as 3E5, 6B9, 4C2, 4B7. Subtype analysis revealed that mAbs 3E5, 4C2 and 4B7 against SARS-CoV N protein belong to IgG₁ while 6B9 is allied to IgG_2a. Western blot analysis showed that the four mAbs could react with SARS-CoV N protein but not with N protein of infectious bronchitis virus.更多还原