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SARS-冠状病毒(SARS-CoV)棘突蛋白(Spike,S)是构成病毒包膜突起的主要成分,属于型膜糖蛋白,全长1255aa,在病毒入侵宿主细胞中起重要作用.本文利用生物信息学技术,界定冠状病毒SARS-CoVS蛋白的抗原决定区域,通过PCR扩增相应片段,克隆至原核表达载体pET-H,得到四个重组质粒pET-HS1、pET-HS3、pET-HS4、pET-HS5.经序列测定证实后,转化大肠杆菌BL21(DE3),IPTG诱导获得表达,产物经紫外薄层扫描显示目的蛋白占菌体细胞总蛋白量的20%~30%.经Westernblot检测,表达蛋白与恢复期SARS病人血清呈高反应原性.纯化相应蛋白免疫小鼠,所获抗血清效价达1∶3×103.与SARS诊断试剂反应呈阳性,这一工作将为进一步研究S蛋白的定位、功能及SARS诊断提供帮助.
SARS-CoV Spike (S) is the main component that forms the envelope of the virus envelope and belongs to the type of glycoprotein with a total length of 1255aa, which plays an important role in virus invasion of host cells.In this paper, According to the information technology, the antigenic region of coronavirus SARS-CoVS protein was defined. The corresponding fragment was amplified by PCR and cloned into the prokaryotic expression vector pET-H to obtain four recombinant plasmids pET-HS1, pET-HS3, pET- -HS5. After confirmed by sequence analysis, E. coli BL21 (DE3) was transformed into E.coli BL21 (DE3) and induced by IPTG, and the product showed 20% -30% of the total protein of mycelium by UV scanning. The protein was highly reactive with the serum of convalescent SARS patients, and the titer of antiserum was 1: 3 × 103 purified from the corresponding protein-immunized mice.The result was positive with SARS diagnostic reagent.This work will be to further study the S protein Positioning, function and diagnosis of SARS help.