应用噬菌体随机肽库技术筛选戊型肝炎病毒中和表位模拟肽  

Selection of a Peptide Mimic the Neutralization Epitope of Hepatitis E Virus with Phage Peptide Display Technology

作　　者：顾颖[1] 张军[1] 王颖彬[1] 李少伟[1] 杨海杰[1] 罗文新[1] 夏宁邵[1] 

机构地区：[1]厦门大学细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005

出　　处：《生物工程学报》2003年第6期680-685,共6页Chinese Journal of Biotechnology

基　　金：福建省科技重大项目 (No .2 0 0 2F0 13 );教育部跨世纪优秀人才培养计划~~

摘　　要：以识别戊型肝炎病毒 (HEV)构象依赖性中和表位的单克隆抗体 8C11、8H3作为固相筛选分子 ,对噬菌体随机 7肽库进行 4轮筛选后 ,随机挑取单克隆噬菌体进行测序。合成优势 7肽序列基因 ,将其插入HBcAgAA78 83位置之中 ,进行原核表达 ,所获重组蛋白经蛋白印迹实验证实可与相应单抗结合 ,电镜下可见重组蛋白能形成与HBcAg相似的类病毒颗粒。化学合成单抗 8H3筛选出的优势 7肽 ,所获 7肽经生物传感器结合实验证实与单抗8H3结合。这些结果提示用噬菌体 7肽库可以筛选出部分模拟构象性表位的短肽 ,为亚单位疫苗的研制提供了新的思路。Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3′-polyadenylated RNA about 7 5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2,ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N′-His-Pro-Thr-Leu-Leu-Arg-Ile-C′, named 8C11A) and 8H3 (N′-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C′, named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E.coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but

关 键 词：戊型肝炎病毒 中和表位 噬菌体肽库 表位模拟肽 

分 类 号：R346[医药卫生—基础医学]