摘要
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I型膜糖蛋白,全长1255 aa,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-CoV S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-H S1、pET-H S3、pET-H S4、pET-H S5.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Western blot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.
According to the results of bioinformatics analysis, the epitopes of S protein are conformed. The fragments of Spike protein gene was frist obtained by polymerase chain reaction, subsequently cloned into pet-H vector, and produced the recombinant plasmid pET-H S1, pET-H S3, pET-H S4 and pET-H S5. The recombinant plasmids were transformed into E. coli BL21 （DE3）. After the strains with recombiment plasmid are induced by IPTG, the expression level was about 20%-30% respectively. Western-blot analysis demonstrated that the recombinant proteins strongly could react with SARS positive sera of recovered patients but not with normal sera tested. Meanwhile, BALB/c mice were immunized by every recombinant S protein expressed in E. coli respectively, and the neutralizing titer of anti-serum was 1 ： 3 × 10^3 and exhibited a positive reaction with diagnosis agents. These results may be of help for further experiment study on SARS.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第3期73-77,共5页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
国家重点基础研究发展计划(973计划)资助项目(2003CB514101)
天津市科委应急攻关计划资助项目(033100211)
