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目的筛选癌胚抗原含T、B细胞表位的短肽并分析其诱导机体产生体液免疫的特性。方法利用免疫信息学方法预测CEA CTL表位,结合我们早期对CEA B表位预测的结果,选择含多个CTL表位肽和B表位肽的CEA氨基酸序列580～598处短肽作为目的基因。目的基因密码子优化后采用pET32a(+)原核表达系统表达融合蛋白,经SDS-PAGE和Western blot-ting鉴定融合蛋白的抗原性;经镍螯合亲和层析柱(Ni-NTA Agarose)纯化CEA580～598全长融合蛋白,纯化蛋白用佐剂乳化后经日本大耳白家兔背部皮下多点免疫注射,采用Western blotting检测血清抗体的特异性,采用ELISA法检测血清抗体水平及变化趋势。结果含多个T、B细胞表位的短CEA580～598在大肠杆菌中获得了高效表达,表达量占总蛋白的25.6%;表达产物的相对分子质量(Mr)约21kDa,与预期Mr相符;Western blotting分析结果显示,在21kDa处出现特异性条带。日本大耳白兔经CEA580～598融合蛋白免疫后产生高水平的特异性抗体,与空载体和PBS对照组比较具有显著性差异(P<0.05),制备的免疫血清不但能特异识别CEA580～598短肽,而且能特异识别天然CEA抗原。结论 CEA580～598短肽融合蛋白具有较强的免疫原性,诱导的兔免疫血清能特异性结合CEA抗原,为基于CEA表位疫苗的研究奠定理论和实验基础。
Objective To screen short peptides containing T and B cell epitopes of carcinoembryonic antigen and analyze their characteristics of inducing humoral immunity. Methods The CEA CTL epitopes were predicted by immunological informatics. Based on our previous prediction of CEA B epitopes, we selected 580-598 amino acids of the CEA epitope peptide and B epitope peptide as the target genes . After optimization of codon usage of the target gene, the fusion protein was expressed by using the prokaryotic expression system pET32a (+), and the antigenicity of the fusion protein was confirmed by SDS-PAGE and Western blot-ting. The fusion protein was purified by Ni-NTA Agarose affinity chromatography CEA580 ~ 598 full-length fusion protein, purified protein was emulsified with adjuvant, and then subcutaneously injected into the back of white rabbits. The specificity of serum antibody was detected by Western blotting. The level of serum antibody and the change trend of serum were detected by ELISA. Results The short CEA580 ~ 598 with multiple T and B cell epitopes was highly expressed in E. coli and expressed 25.6% of the total protein. The relative molecular mass (Mr) of the expressed product was about 21 kDa, which was consistent with the expected Mr. Western blotting analysis showed that a specific band appeared at 21 kDa. The results showed that CEA580 ~ 598 fusion protein produced high level of specific antibody in Japanese white rabbits, which was significantly different from that in empty vector and PBS control group (P <0.05). The prepared immune serum could not only recognize CEA580 ~ 598 Short peptides, but also specifically recognize native CEA antigens. Conclusion CEA580 ~ 598 peptide fusion protein has strong immunogenicity. The induced rabbit serum can specifically bind with CEA antigen, laying a theoretical and experimental foundation for the study of CEA epitope vaccine.