SARS-CoV核衣壳蛋白的体外表达及单克隆抗体功能鉴定  

In vitro expression of SARS coronavirus (SARS-CoV) nucleocapsid protein and examination of the function of monoclonal antibody induced by the expressed protein

作　　者：陈洪勋[1] 严兴成[2] 应建跃[2] 吴建祥[1] 周继勇[1] 

机构地区：[1]浙江大学动物科学学院 [2]浙江大学校医院华家池分院,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2005年第2期232-236,共5页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30230270).

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.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 IgG_1 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.

关 键 词：严重急性呼吸综合征(SARS) 冠状病毒 N蛋白 单克隆抗体 

分 类 号：R373.9[医药卫生—病原生物学] R392.11[医药卫生—基础医学]