Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology.
OBJECTIVE
To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3.


METHODS
8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4 rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E.coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor.


RESULTS
Twenty-one positive monoclonal phages (10 for 8C11, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 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 the HBcAg vector, then expressed in E.coli. The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemo-synthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor.


CONCLUSIONS
These results implicate that conformational neutralizing epitope can be partially modeled by a short peptide, which provides a feasible route for subunit vaccine development.
Gu
Ying
Y
The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, Fujian Province, China.
Zhang
Jun
J
Wang
Ying-Bing
YB
Li
Shao-Wei
SW
Yang
Hai-Jie
HJ
Luo
Wen-Xin
WX
Xia
Ning-Shao
NS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D000939:Epitopes / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006512:Hepatitis B Core Antigens / Q000235:genetics; D016752:Hepatitis E virus / Q000235:genetics* / Q000276:immunology* / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D019151:Peptide Library; D011487:Protein Conformation; D014761:Viral Hepatitis Vaccines / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D014771:Virion / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006512:Hepatitis B Core Antigens; D019151:Peptide Library; D014761:Viral Hepatitis Vaccines