腺病毒六邻体蛋白型间线性化抗原位点的研究

Characterization of intertype specific epitopes on adenoviruses hexon

目的 对人类腺病毒 (adenovirus,AdV)六邻体型间抗原位点的特性进行研究。方法 通过计算机对腺病毒六邻体氨基酸序列进行比较分析 ,结合抗原性的预测结果和六邻体蛋白三维空间结构的肽段暴露状态 ,选定了保守性抗原位点进行多肽合成或通过构建重组质粒表达蛋白 ,将合成的六邻体肽段和表达纯化的蛋白抗原免疫动物后 ,用免疫印迹和间接免疫荧光方法检测抗血清的免疫特异性。结果 免疫印迹分析显示 ,抗多肽抗体和抗重组蛋白抗体均与腺病毒的六邻体蛋白特异性识别。间接免疫荧光显示 ,腺病毒感染HeLa细胞核内荧光着色 ,并且抗血清有较好的腺病毒型间反应性。合成多肽抗体与含有相同肽段的重组蛋白抗原产生特异性结合。结论 在腺病毒六邻体蛋白中存在有线性化的型间抗原位点 。

Objective To characterize the intertype epitopes on human adenovirus (HAdV) hexon.Methods Based on computerized analysis on adenoviruses sequence of genomic alignment,antigenicity prediction and 3-D structure characteristics of hexon subunit,several peptides of hexon of adenoviruses were chosen to be synthesized or recombinant proteins of the hexon were expressed in %E.coli% by use of PGEX-5X. To identify the existence of intertype epitopes,the antisera raised with synthetic peptides or purified recombinant proteins were analyzed with Western blot and immunofluorescent assay.Results The results of Western blot indicated that both peptide and recombinant antibodies showed specific reactivities with hexons of HAdV-3,4,7 individually. Meanwhile,typical stain of immunofluorescence was found on HeLa cells infected with these HAdV by incubation with peptide as well as recombinant antibodies. Also,antibodies raised against peptide recognized the recombinant hexon protein in which a corresponding region of peptides was covered.Conclusion Most of the predicted intertype epitopes of HAdV hexon were exclusively found in synthetic peptides and recombinant proteins. These intertype epitopes showed to be continuous and sequential which could be employed for development of antibodies of diagnostic use.