【摘要】 目的:以戊型肝炎病毒(HEV)ORF2编码的重组蛋白p166为例,研究蛋白标签GST对融合表达的重组蛋白抗原结构的影响。方法:以HEV中国株重组蛋白p166Chn-GST为免疫原,制备单克隆抗体(mAb),与代表HEV4个基因型的摩洛哥株、墨西哥株、美国株和中国株p166的GST或His融合蛋白、中国株非融合重组蛋白p179Chn以及GST融合的HEV无关蛋白进行ELISA检测,鉴定mAb所识别的抗原表位。结果:获得3株稳定分泌抗p166Chn-GST的杂交瘤细胞株,分泌的mAb1A8、9B4和8H10与p166Chn-GST反应,与GST不反应。其中1A8和9B4可与带GST标签的4种p166-GST蛋白以及N和C端截短的p146Chn-GST、p137Chn-GST反应,而不与4种p166-His蛋白反应,也不与p179Chn反应,与HEV病毒颗粒竞争试验阴性,与GST融合的HEV无关蛋白无交叉反应性,表明1A8和9B4识别的抗原表位不是HEV病毒颗粒表面天然存在的抗原表位,而是GST与HEV ORF2编码蛋白的465-601aa区段序列共同形成的新的抗原表位。结论:GST能够赋予基因工程重组蛋白以新的抗原特性,它与融合表达的重组蛋白可以共同形成新的抗原表位。更多还原

【Abstract】 AIM: To investigate the effect of a GST tag on the antigenic structure of GST fusion-expressed and ORF2-encoded recombinant proteins of hepatitis E virus (HEV). METHODS: The monoclonal antibodies (mAb) were prepared with a GST fusion protein, p166Chn-GST, which was derived from a Chinese HEV strain. Then they were tested by indirect ELISA, competition ELISA and Western blot with different GST fusion, His fusion or non-fusion recombinant proteins derived from HEV reference strains of all 4 genotypes and other non-HEV recombinant proteins. RESULTS: Three mAb named 1A8, 9B4 and 8H10 were obtained. All of them reacted to p166Chn-GST but did not react to GST. mAb 1A8 and 9B4 reacted to 4 p166-GST proteins of different HEV genotypes and 2 N-or C-terminal truncated p166Chn-GST proteins named p146Chn-GST and p137Chn-GST, but they did not react to 4 p166-His proteins of different HEV genotypes and a non-fusion p179Chn protein. No detectable signals were found when 1A8 and 9B4 were subjected to HEV antigen competition ELISA or Western blot after SDS-PAGE. No cross reaction was observed between the two mAb and HEV-irrelevant GST fusion proteins, either. CONCLUSION: A novel antigenic epitope recognized by mAb 1A8 and 9B4 appears on the GST fusion-expressed and ORF2-encoded HEV recombinant proteins and it is dependent on the conformational folding of both GST and HEV sequences.更多还原