Generation Of Monoclonal Antibody To Porcine Rotavirus VP7Protein And Identification Of Its Mimic Epitope

Porcine Rotavirus (PRV) belongs to the family Reoviridae and is one of the causative agents of viral diarrhea in young children and animals. PRV is popular in a wide range around the world. The piglets weaned newly are most vulnerable, the main clinical symptom is more severe diarrhea. PRV make piglets vomiting, diarrhea, dehydration, acid-base imbalance. PRV infection of piglets mixed with other secondary bacterial infection caused increasing mortality in the pig industry, and great economic losses happened. Considering the outer capsid glycoprotein VP7as a very important protein of PRV, it was undertaken to prepare the anti-PRV VP7monoclonal antibody and character its mimic epitope. The results are as followed:In this study, the positive plasmid pET-30a-VP7was transformed into E.coli BL21, after induction and expression with IPTG, the recombinant protein of37kDa molecular size was primarily in the form of inclusion bodies. Western-blot verified porcine rotavirus VP7protein has high immunogenicity. Vaccination of BALB/c mice with purified VP7protein, the immunized murine spleen cells and myeloma SP2/0cell were fused. By indirect ELISA, the positive clones were screened out, and then positive wells were subcloned by limiting dilution and got porcine rotavirus VP7monoclonal antibody (MAb)3B6. Subclass antibody test confirmed that MAb3B6belongs to IgG2b subclass. Indirect immunofluorescence (IFA) and Western blot test (Western blot) showed that, MAb3B6could react with porcine rotavirus VP7protein and PRV. In the ELISA assay, MAb3B6could specifically differentiate PRV from other viruses.Phage display technique was widely used in antigen identification, epitope screening, receptor and functional ligand characterization and other aspects. Subsequently, purified ascites containing MAb3B6was used as an immobilized target in a subtract biopanning process using a12-mer phage display random peptide library, using a packet in descending order of antibody concentration and binding time, and gradually increase the concentration of Tween-20washing solution, and the volume kept substantially constant input phage selection methods to improve the strength, so as to obtain high affinity phage with specificity. After four rounds of pannings, ten phages bearing the consensus peptide VPLGTDNGDIWV and having a specific binding activity to the target MAb were identified. Sequence alignment between phage-displayed12-mer peptide and VP7protein suggested that a potential motif that is identical with167(P),178(T),179(D) or184(W) aa in the PRV VP7protein. Competition ELISA showed that the identified phage reduced the interaction of PRV with anti-VP7MAb and there was a positive correlation between the reduction effect and the dose of peptides. The four amino acid motif (P-TD-W) functions as a spatial conformation epitope of PRV VP7protein.