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目的:预测及鉴定尤文肉瘤EWS-FLI1融合蛋白的B淋巴细胞表位。方法:采用综合法预测EWS-FLI1蛋白的二级结构及B淋巴细胞表位,运用标准Fmoc方案合成预测的表位肽,HPLC和MS进行表位肽的纯度分析及分子量鉴定,ELISA法检测表位肽的抗原性,并测定表位肽特异性免疫血清效价,Western blot鉴定免疫血清与EWS-FLI1蛋白亲合力。结果:通过综合法预测得到3个高分值的B淋巴细胞表位,HPLC分析合成的表位肽纯度>85%,MS鉴定表位肽的分子量无误,ELISA法检测证实3个B淋巴细胞表位肽均可获得强的抗原抗体反应,其中表位肽P2的抗原性最强,在1∶40时A450=2.46,达到最高,1∶10 240稀释后抗原抗体反应仍呈阳性;用这3个B淋巴细胞表位肽免疫新西兰兔也能获得理想的抗体效价,其中表位肽P2获得的抗体效价最高,1∶512 000稀释时A450=1.11;Western blot鉴定表位肽P1、P2免疫血清能够结合EWS-FLI1蛋白。结论:尤文肉瘤EWS-FLI1蛋白的B淋巴细胞表位肽P1、P2具有潜在的抗原性和免疫原性。
Objective: To predict and identify B lymphocyte epitopes of Ewing’s sarcoma EWS-FLI1 fusion protein. Methods: The secondary structure of EWS-FLI1 protein and epitopes of B lymphocytes were predicted by the integrated method. The predicted epitope peptides were synthesized by standard Fmoc protocol. The purity and molecular weight of epitope peptides were determined by HPLC and MS. The specificity of the peptides was determined. The titer of the epitope-specific immunized sera was determined. The affinity of the immune serum and EWS-FLI1 protein was identified by Western blot. Results: Three high-value B lymphocyte epitopes were predicted by the synthetic method. The purity of the epitope peptides synthesized by HPLC was higher than 85%. The molecular weight of the peptide was the same as that of the MS. The results of ELISA showed that the three B lymphocyte epitopes Peptide can obtain a strong antigen-antibody reaction, epitope peptide P2 antigenic strongest, at 1:40 when A450 = 2.46, reaching the highest, 1:10 240 dilution of the antigen-antibody reaction was still positive; B lymphocyte epitope peptide immunized New Zealand rabbits can obtain the ideal antibody titer, epitope peptide P2 obtained the highest antibody titer, A450 = 1.11 at 1: 51200 dilution; Western blot identification epitope peptide P1, P2 Immune serum is able to bind EWS-FLI1 protein. Conclusion: Ewing-sarcoma EWS-FLI1 protein B lymphocyte epitope peptides P1, P2 have potential antigenicity and immunogenicity.