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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。
OBJECTIVE: To investigate the effect of protein tag GST on the fusion protein antigenic structure of recombinant protein p166 encoded by the hepatitis E virus (HEV) ORF2. METHODS: Monoclonal antibody (mAb) was prepared with HEV Chinese strain recombinant protein p166Chn-GST as the immunogen, and mixed with GST or His fusion protein of the Moroccan strain, Mexican strain, American strain and Chinese strain p166 representing HEV genotypes. The Chinese Strain non-fusion recombinant protein p179Chn and GST-fused HEV unrelated protein were detected by ELISA to identify the antigen epitope recognized by mAb. Results: Three hybridoma cell lines stably secreting anti-p166Chn-GST were obtained. The secreted mAb1A8, 9B4 and 8H10 reacted with p166Chn-GST and did not react with GST. 1A8 and 9B4 could react with four kinds of p166-GST proteins with GST tag and p146Chn-GST and p137Chn-GST with N and C-terminal truncation without reacting with four p166-His proteins and with p179Chn, Negative competition with HEV virus particles and no cross-reactivity with HEV-associated proteins fused to GST suggested that the epitopes identified by 1A8 and 9B4 were not naturally occurring epitopes on the surface of HEV virions, but that GST and HEV ORF2 encoded proteins 465-601 aa region sequence together form a new antigenic epitope. CONCLUSION: GST can confer new antigenic characteristics to the recombinant protein, which together with the recombinant protein can form a new antigenic epitope.