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目的 克隆和表达SARS冠状病毒N蛋白 ,并分析其免疫学活性。方法 采用RT PCR从SARS冠状病毒RNA中扩增出编码N蛋白的基因 ;经克隆和测序分析后 ,亚克隆至表达载体pGEX 4T 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定 ;阳性菌株经IPTG诱导 ,SDS PAGE分析 ;大量诱导表达N蛋白 ,亲和层析予以纯化 ;免疫印迹分析纯化蛋白对SARS的诊断效果 ;用纯化的融合蛋白免疫小鼠观察其诱导的抗体应答。结果 RT PCR扩增出N蛋白基因的特异片段 ,获得的阳性克隆序列与Gen Bank中登录的SARS冠状病毒的N蛋白基因序列同源性为 99.8% ;N蛋白基因被亚克隆到表达载体pGEX 4T 2 ,在JM10 9中表达了N蛋白 ,表达的蛋白经亲和层析获得纯化 ;纯化蛋白能被SARS病人血清识别 ;免疫小鼠诱导产生了高滴度的抗体。结论 成功构建了SARS冠状病毒N蛋白的重组表达质粒 ,在大肠杆菌中表达的N蛋白融合蛋白具有良好的免疫学活性。
Objective To clone and express SARS - CoV N protein and analyze its immunological activity. Methods The gene encoding N protein was amplified from RNA of SARS coronavirus by RT PCR. After cloning and sequencing, it was subcloned into expression vector pGEX 4T 2 and transformed into E. coli JM109. PCR and restriction enzyme digestion were performed. The positive strains Induced by IPTG and analyzed by SDS PAGE. N protein was induced in large quantity and purified by affinity chromatography. The purified protein was analyzed by immunoblot for the diagnosis of SARS. The immunized mice were immunized with the purified fusion protein to observe the induced antibody response. Results The specific fragment of N protein gene was amplified by RT-PCR. The positive cloned sequence was 99.8% identical to the N protein gene of SARS coronavirus registered in Gen Bank. The N protein gene was subcloned into expression vector pGEX 4T The N protein was expressed in JM109, and the expressed protein was purified by affinity chromatography. The purified protein could be recognized by the serum of SARS patients. The immunized mice induced high titer antibody. Conclusion The recombinant expression plasmid of SARS-CoV N protein was successfully constructed. The N protein fusion protein expressed in E. coli has good immunological activity.