SARS-CoV S蛋白抗原表位富集区的表达与初步应用

Expression and preliminary application of the immunodominant region of SARS-CoV spike protein

扩增并克隆编码SARS-CoV S蛋白中包含多个抗原表位的第539～630氨基酸残基区域的基因片段。基因片段经酶切处理后插入表达载体pGEX-6p-1的BamHⅠ和XhoⅠ位点之间,构建成融合表达质粒,转化宿主菌BL21经IPTG诱导后融合基因得到了表达,表达产物可用谷胱甘肽sepharose 4B RediPack亲和层析柱纯化。用5份SARS患者康复期血清对表达融合蛋白进行ELISA和免疫印迹试验,分析融合蛋白的免疫反应性,结果表明重组融合抗原在ELISA和免疫印迹试验中与5份SARS患者康复期血清均具有良好的免疫反应性。试验结果提示该SARS-CoV S蛋白第539～630氨基酸残基区域可作为SARS的诊断抗原。

In this study, the gene fragment enconding 539 to 630 amino acid resides of SARS-CoV spike protein was PCR amplified and inserted into the expression vector pGEX-6p-1 between the restriction enzyme sites BamH Ⅰ and Xho Ⅰ . The resultant plasmid was transformed into E.coli BL21 host cell and induced with IPTG. SDS-PAGE analysis showed that the recombinant protein was expressed in a soluble form with a molecule weight about 37 Ku, The protein was purified and used as antigen in ELISA and Western blot for detection of SARS-CoV spike protein specific antibody. The immunological properties of the recombinant protein were evaluated against five SARS convalescent human sera. The results showed that the recombinant protein reacted positively with all five SARS convalescent human sera but not with normal human sera. This work demonstrated that the fragment of S 539-630 could be an effective antigen candidate for SARS diagnosis and may be useful in the development of diagnostic reagents and genetic engineering vaccines.