人感染 H7 N9禽流感病毒血凝素蛋白线性 B 淋巴细胞抗原表位的预测及鉴定

Prediction and identification of linear B-cell epitopes in the hemagglutinin of H7N9 avian influenza virus in human

目的：预测并鉴定人感染H7N9禽流感病毒血凝素（HA）蛋白的线性B淋巴细胞抗原表位及H7亚型的特异性。方法选取绍兴市人民医院提供的人感染H7 N9患者不同时间的三份血清样本和一份健康对照血清样本，采用TMHMM Sever v．2．0预测蛋白质胞外区，采用DNAStar Lasergene’ s Protean、BcePred和ABCpred预测其优势B淋巴细胞抗原表位，并用间接酶联免疫吸附试验（ ELISA）验证预测的B淋巴细胞抗原表位的免疫原性。使用Clustal X2．1软件将预测抗原表位与GenBank上H7N9和H1～H16亚型流感病毒HA蛋白的氨基酸序列进行多重序列比对以分析预测抗原表位的H7亚型特异性，采用Cn3D 4．3．1软件分析预测的线性B淋巴细胞抗原表位在H7N9禽流感病毒HA蛋白中的分布情况。结果 HA蛋白可能的B淋巴细胞表位位于胞外区N端的第172～183、363～380、452～472和第491～506四个区段。 ELISA结果表明，四个预测的B淋巴细胞表位均与已确诊患者血清发生阳性反应。多重序列比对结果发现第172～183和452～472区段与其他亚型氨基酸序列差异最大，可能具有更好的H7亚型特异性。 HA蛋白的三维结构进一步证明预测的四个B淋巴细胞抗原表位均位于HA蛋白表面。结论鉴定出了四个 HA蛋白线性B淋巴细胞抗原表位，其中第172～183和452～472区段具有更好的H7亚型特异性，可作为新型疫苗表位设计和H7抗原和抗体的特异性检测的依据。

Objective To predict and identify liner B-cell epitopes in the hemagglutinin （ HA） of human-infected avian-origin H7N9 influenza virus and analyze the specificity of H7 subtype.Methods Three serum samples collected at different times from the same patient who was confirmed to be infected with H7N9 influenza virus were provided by Shaoxing People’s Hospital, and one serum sample from healthy person was collected as the control.The extracellular region of HA protein was predicted by TMHMM Sever v.2.0.The potential B-cell epitopes were predicted by DNAStar Lasergene’ s Protean, BcePred and＆amp;nbsp;ABCpred tools, and the immunogenicity of the predicted B cell antigen epitopes was assessed by indirect enzyme-linked immunosordent assay （ ELISA ） .H7 subtype specificity was analyzed by comparing HA protein amino acid sequence with H7N9 and H1-H16 subtype influenza virus from Genbank using Clustal X 2.1 software, and Cn3D 4.3.1 software was used to detect the distribution and 3D structure of predicted epitopes on the HA protein of H7N9.Results The potential B-cell epitopes may be located in 172-183, 363-380, 452-472 and 491-506 of extracellular N-terminus of HA protein.ELISA showed that four predicted eptiopes specifically reacted with positive serums from patient.Multi-sequence alignment demonstrated that peptide 172-183 and 363-380 had higher H7 subtype specificity compared with amino acid sequences of other subtypes.Moreover, the predicted linear B-cell epitopes all located on the surface of HA protein according to the 3D structure analysis.Conclusion Four potential B-cell epitopes were identified, in which peptide 172-183 and 363-380 have higher H7 subtype specificity, and may be used in the design of epitope-based vaccines and diagnostics tests.