论文部分内容阅读
目的研究SARS冠状病毒E蛋白对SARS诊断和预防的价值,利用原核表达系统克隆和表达E蛋白并纯化。方法采用RT-PCR从SARS冠状病毒RNA中扩增出编码E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BL21(DE3),PCR和双酶切鉴定;阳性菌株经IPTG诱导,SDS-PAGE分析,进一步以SARS病人血清进行免疫印迹分析;大量诱导表达E蛋白,亲和层析予以纯化。结果RT-PCR扩增出E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E蛋白基因序列同源性为100%;E蛋白基因被亚克隆至表达载体pGEX-4T-2,在BL21中获得表达,表达产物能被SARS病人血清识别。表达的E蛋白经亲和层析获得纯化。结论成功构建了SARS冠状病毒E蛋白的重组表达质粒,在大肠杆菌中表达的E蛋白的融合蛋白具免疫活性。
Objective To study the value of SARS-CoV E protein in the diagnosis and prevention of SARS. The prokaryotic expression system was used to clone and express E protein. Methods The gene encoding E protein was amplified by RT-PCR from SARS coronavirus RNA. After cloning and sequencing analysis, it was subcloned into expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) The positive strains were induced by IPTG and analyzed by SDS-PAGE. Western blot analysis was further performed on the serums of SARS patients. E protein was induced in large quantities and purified by affinity chromatography. Results The specific fragment of E protein gene was amplified by RT-PCR. The positive cloned sequence was 100% identical to the E protein gene of SARS coronavirus registered in Gen Bank. The E protein gene was subcloned into the expression vector pGEX -4T-2, expressed in BL21, the expression product can be recognized by the serum of SARS patients. The expressed E protein was purified by affinity chromatography. Conclusion The recombinant expression plasmid of SARS-CoV E protein was successfully constructed. The fusion protein of E protein expressed in E. coli was immunocompetent.