关键词: 猪流行性腹泻病毒, S2基因, B细胞表位, 单克隆抗体

Abstract:

[Objective] To identify the B-cell epitope peptide of porcine epidemic diarrhea virus （PEDV） S2 gene by combinative use of bioinformatics software and monoclonal antibody technology. [Method] The B-cell epitope of PEDV S2 gene was screened using CLC Sequence viewer 6.8 software and IEDB online database, and the obtained epitope peptide was synthesized artificially. Female BALB/c mice were immunized with the conjugate of epitope peptide and keyhole hemocyanin （KLH） as antigen. Mice with higher antibody titers were identified by ELISA assay and then received an additional immunization. The spleen of the mice was taken 3 days post immunization to prepare the splenocyte suspension for cell fusion. The cells were grown on HAT selective medium to screen for effective hybridoma cells. The positive clones screened by ELISA assay were then used for expanding culture. Positive hybridoma cells were intraperitoneally injected to mice and ascites were collected. ELISA assay was used to determine the antibody titers in mice ascites and in the supernatants of monoclonal cell strains. The cells with the highest antibody titers was used as cell strain for subsequent use. [Result] The selected B-cell epitope peptide sequence was MQYVYTPTYYML. Following immunization with the peptide antigen, the serum antibody titer before cell fusion reached 1:2 000. The ELISA assay of ascites from BALB/c mice and the supernatants from monoclonal cell strain cultures demonstrated that the antibody titer reached 1:4 000. [Conclusion] The B-cell epitope of PEDV S2 gene was identified, which may be helpful for the vector construction of a epitope based peptide vaccine against PEDV.

Key words: porcine epidemic diarrhea virus, S2 gene, B-cell epitope, monoclonal antibody