用抗合成多肽抗体分析呼吸道HAdV六邻体保守区抗原表位

Delineation of epitopes in conserved regions of respiratory HAdV hexons using anti-peptide antibodies

目的 分析人类腺病毒 (HAdV)六邻体保守区氨基酸序列 ,合成保守区多肽并制备抗多肽抗体 ,以确定型间共同的特异性表位。方法 用ClustalX、Antheprot等软件 ,对HAdV六邻体进行分析 ,确定保守区并分析其抗原性。合成抗原性多肽 ,免疫家兔制备抗肽多克隆抗体。用间接免疫荧光法和Westernblot法 ,检测所制备的抗肽抗体与病毒抗原的反应性。结果 根据HAdV 2六邻体的保守区合成 9种肽段 ,分成 3组免疫家兔 ,共获得 3组抗肽抗体。所有的抗肽抗体在 1∶10 0 0稀释度时 ,与 3个型别的HAdV感染细胞裂解物中相对分子质量 (Mr)约为 116 0 0 0的六邻体亚单位具有特异性反应。抗肽抗体对HAdV感染细胞有特征性荧光染色。 3组抗肽抗体中 ,有 7个抗体与 3个型别的HAdV感染细胞均具有阳性染色反应。结论 合成的保守区多肽能够诱导抗肽抗体产生 ,诱导产生的抗体与HAdV 3、 4、 7具有免疫反应性。

Aim To analyze conserved region amino acid sequences of human adenovirus (HAdV) hexon, and to synthesize conserved peptides and prepare anti-peptide antibodies, then to determine intertype-specific epitopes. Methods HAdV hexons were analyzed with ClustalX and Antheprot softwares, so as to determine conserved regions and analyze their antigenicity. Conserved peptides were synthesized and rabbits were immunized with the synthetic peptides to prepare anti-peptide antibodies. The reactivities between anti-peptide antibodies and HAdV were determined by indirect immunofluorescent assay (IFA) and Western blot. Results According to conserved regions of HAdV-2 hexon, 9 peptides were selected and subdivided into three groups to immunize rabbits, obtaining three groups of anti-peptide antibodies. All anti-peptide antibodies at a dilution of 1∶1 000 reacted specifically with hexon subunit of relative molecular mass (Mr) 116 000 in lysates of HAdV-3, -4, -7-infected cells. Characteristic fluorescence in virus-infected cells was shown with anti-peptide antibodies. Of the three group anti-peptide antibodies, 7 antibodies could crossreact to all three types of HAdV by IFA. Conclusion The synthesized conserved region peptides can elicit the production of anti-peptide antibodies and these antibodies can crossreact to HAdV-3, -4, -7. It is feasible to predict epitopes by software analysis and then prepare anti-peptide antibodies.