[Selection of a peptide mimic the neutralization epitope of hepatitis E virus with phage peptide display technology].
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 only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development.
Gu
Ying
Y
Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China.
Zhang
Jun
J
Wang
Ying-Bin
YB
Li
Shao-Wei
SW
Yang
Hai-Jie
HJ
Luo
Wen-Xin
WX
Xia
Ning-Shao
NS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000378:metabolism; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006512:Hepatitis B Core Antigens; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins