Abstract:

The non-structural protein p7 of bovine viral diarrhea virus（BVDV）is called viroporin due to its ion channel activity,which participates in the process of virus entry,release and replication. To further investigate the mechanism of p7 protein forming ion channel,p7 specific polyclonal antibody was prepared using the synthetic peptide. According to the amino acid information of NADL p7 of BVDV strain in GenBank,the bioinformatics analysis was conducted,and the surface antigen polypeptide was synthesized and conjugated with the protein of keyhole limpet hemocyanin（KLH）served as the carrier protein. The protein was purified by reversed-phase-high performance liquid chromatography（RP-HPLC）,the purity was analysed,and qualitative identification was conducted by mass spectrometry. Then the peptide plus Freund’s adjuvant was emulsificated and multiple subcutaneously injected into the New Zealand white rabbit,and the immune dose was 400 μg per rabbit,which repeated continuously 5 times. The serum was purified by peptide affinity column at 10 d after immunization,and the polypeptide polyclonal antibody was acquired. Indirect ELISA and SDS-PAGE were applied to detect the titer and purity of the polyclonal antibody. Immunofluorescence staining was used to detect the reactivity and specificity of the polyclonal antibody after BVDV NADL infected the MDBK of bovine renal cells. Bioinformatics analysis showed that the main antigenic epitopes were located in the N-terminal of p7 protein. The peptide MSQYGAGEIVMMGN-Cys-KLH was synthesized after KLH conjugation. The purity of the peptide reached 80.19% and the molecular weight was 794.2 Da. After 5 times of immunization and the serum was purified using the peptide affinity column,the titer of the polyclonal antibody detected by indirect ELISA was 1:100 000,and the antibody purity detected by Western blot was 90.2%. Immunofluorescence staining showed that the staining of the polyclonal antibody was positive on the membrane of MDBK cells infected with BVDV NADL,which was consistent with the location of p7 ion channel. The rabbit anti-BVDV p7 polypeptide polyclonal antibody was successfully prepared,and had high reactivity and specificity,which provides a useful tool for further investigation on the mechanism of p7 forming ion channels.

Key words: bovine viral diarrhea virus, p7 protein, ion channel protein, polypeptide, polyclonal antibody