Capsid protein antigen epitope prediction and monoclonal antibody preparation for Norovirus GⅡ

Objective To express the noruvirus capsid protein(VP1), prepare monoclonal antibodies against it and understand the noruvirus capsid proteins epitope characteristics. Methods PCR amplification of VP1 gene of Norovirus GⅡ(Huzhou strains ) was conducted, the clone of target gene to the vector pET28a(+) and transferring into E. coli were conducted too. BALB/c mice were immunized with purified expression product. The spleen cells of successfully immunized mice were fused with myeloma cells SP2/0. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the SEPPA for predicting the possible spatial epitopes with the default threshold. Positive clones were screened to obtain monoclonal antibodies corresponding to these epitopes by predicted epitope sequence. Results The recombinant expression vector pET28a(+)-Noro-VP1 was successfully constructed and expressed in E. coli, the recombinant protein-immunized BALB/c mouse spleen cells were fused with SP2/0 cell. The predicted epitope region polypeptide screened ten Hybridoma. Indirect ELISA assay showed that 1E8, 1P10, 2C15 and 2L16 can react with recombinant protein strongly, the other 6 mAbs had weak reaction. Western blotting showed that only 2K10, 1K16, 1E8 can specifically recognized the Norovirus capsid protein expressed in E.coli. Conclusion The mAbs against G Ⅱ norovirus capsid protein was successfully prepared. Epitopes was in capsid protein P2 region. This can be used for the development of rapid immunodiagnostic kits and epitopes research of norovirus capside protein.