SARS-CoV核衣壳蛋白的体外表达及单克隆抗体功能鉴定

为对SARS N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.coli BL21中,IPTG诱导表达,表达产物与兔源GST多抗在66 kD处有很强的反应性,融合蛋白GST-N分别通过GSTrap FF 1 mL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0.85 mg/mL和0.433 mg/mL.利用纯化的GST-N蛋白免疫SPF BALB/c小鼠,获得4株能稳定传代并分泌抗SARS N单克隆抗体(mAb)的杂交瘤细胞,分别命名为3E5、6B9、4C2、4B7,经Western blot分析表明,所获得的4株抗SARS N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.

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

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.