Generation Of Monoclonal Antibody To S_AD Protein Of Porcine Transmissible Gastroenteritis Virus And Identification Of Its Mimic Epitope

Porcine transmissible gastroenteritis virus(TGEV), belongs to the family coronaviridae, is an acute and highly contagious enteric infectious diseases which can be caused by all ages of pigs.TGEV often produces severe vomiting, diarrhea,dehydration and causes great economic loss on the scale of the sucking pig. S protein is a key structural protein of TGEV coronavirus. TGEV S protein is a type I transmembrane protein located on the surface of virus mediating virion binding to specific receptors. There are four antigen epitopes containing A, B, C and D that reside near the N terminal domain. Antigenic sites A and D coding region were defined as SAD which are most critical in inducting neutralizing antibodies. Therefore, the study of TGEV SAD has an important value in porcine transmissible gastroenteritis virus antiviral research.In this study, the positive plasmid SAD was transformed into E.coli BL21. After induction and expression with 37 ℃ over a 7-h period IPTG post-induction, the recombinant protein of 34 kDa molecular size was primarily in the form of inclusion bodies. The resulting SAD protein was used to immunize BALB/c mice followed by the generation of a monoclonal antibody(MAb). Vaccination of BALB/c mice with purified SAD protein, 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 transmissible gastroenteritis virus SAD monoclonal antibody(MAb) 2B10. The immunoreactivity of the MAb to TGEV S protein was confirmed by Western blot analysis. Moreover, immunofluorescence assays showed that the MAb is able to detect cell infection by TGEV. Subclass antibody test confirmed that MAb 2B10 belongs to Ig G2 b subclass and k chain. The MAb achieved in this study can be used as a specific diagnostic reagent for detecting TGEV S protein. In the ELISA assay, MAb 2B10 could specifically differentiate TGEV from other viruses.Ascitic fluid containing anti-TGEV SAD MAb were generated and purified and subsequently used as target for phage display using a packet in descending order of antibody concentration and binding time, and gradually increase the concentration of Tween-20 washing solution, and the volume kept substantially constant input phage selection methods to improve the strength. After 4modified rounds of biopanning, high affinity phage with specificity 10 were sequenced containing five different sequences.Synthetic peptide which has the highest binding active with the virus(SGVYKVAYDWQH and YTSSMLISSSLS),named P-S/P-Y. By evaluating its ability of inhibition virus, we proved the peptide could specific binding with MAb 2B10. q RT-PCR experiments revealed P-Y and P-S could notable reduce the TGEV infections in vitro(p<0.01), the former has a better effect, and peptides inhibited virus in a does-dependent manner.