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汉滩病毒核衣壳蛋白主要B细胞表位的识别及其基因定位 |
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白雪帆 汪毅 潘蕾 黄长形 李光玉 杨为松
【摘要】 目的 确定汉滩病毒核衣壳蛋白(NP)B细胞表位的基因定位。方法 将DNase Ⅰ随机消化的汉滩病毒76/118株S基因片段与丝状噬菌体载体连接后转化MC1061菌，成功构建了汉滩病毒S基因-多肽片段噬菌体肽库。应用12株国产抗汉滩病毒单克隆抗体对表达汉滩病毒NP多肽片段的噬菌体文库进行了3～4轮淘筛(biopanning)，并对阳性噬菌体克隆进行了扩增和测序。结果 确定了S基因产物——病毒核衣壳蛋白氨基端的一个B细胞线性抗原表位，该表位覆盖aa1-aa86的范围，相当于S基因bp37-294，其核心序列可能由aa15-66构成。结论 该线性表位能与多种抗汉滩病毒单克隆抗体发生交叉反应，是汉滩病毒NP的主要抗原表位。
【关键词】 汉滩病毒 抗体，单克隆 噬菌体肽库
The recognition and mapping of epitopes of Hantaan virus nucleocapsid protein by S gene-peptide fragment phage display libraries
BAI Xuefan, WANG Yi, PAN Lei, et al.
Department of Infectious Diseases, Fourth Military Medical University, Xian 710038
【Abstract】 Objective In order to construct the library of Hantaan virus S gene-peptide fragments on phages, screening the epitopes of nucleocapsid protein with monoclonal antibodies against Hantaviruses. Methods Hantaan virus S gene from recombinant plasmid pUC-HTNV-S was digested with DNase Ⅰ and ligated with filamentous phage vector that has been cut with Sfi I. The ligation were transformed into E. coli MC1061 for constructing the phage library of Hantaan virus S gene-peptide fragments. After biopanning with some strains of monoclonal antibodies, the positive phage clones were picked out for sequencing. Results A linear epitope consisting of amino acid (aa) 1-86 within nucleocapsid protein reading frame was determined and its core sequence consists of aa 15-66. Conclusion The linear epitope is a major and cross-reactive antigenic epitope. The findings above are helpful for elucidating the relationship between molecular structure and function of Hantaviruses.
【Key words】 Hantaan virus Antibodies, monoclonal Bacteriophages peptide libraries
核衣壳蛋白(Nucleocapsid Protein, NP)是汉坦病毒(hantaviruses, HV)的主要结构蛋白。已知HV约45000～51000的NP由428～433个氨基酸组成，其N端约100个氨基酸大部分具有亲水性，且能被患者血清及许多单克隆抗体所识别［1-3］。虽然对HV NP氨基端蛋白的原核表达产物的抗原性及免疫原性已进行了许多研究，并已用于出血热的血清学诊断，但该抗原表位的精确一[1] [2] [3] 下一页
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