Preparation Of Monoclonal Antibody To Porcine Epidemic Diarrhea Virus N Protein And Identification Of Its Antigenic Epitopes

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV), is a highly exposed infectious diseases, havinga high mortality rate of suckling piglets. At present, although the commercialization of PEDV vaccine has been widely used, the disease is still prevalent in China, causing serious economic losses. Epidemiological and clinical symptoms of this disease is very similar to the porcine transmissible gastroenteritis and there are difficulties in differential diagnosis and vaccine applicaton, therefore, the development of novel and effective diagnostic methods to PEDV has practical significance.Epitope studies reveal the molecular structure and function of antigen and the antigen-antibody reaction mechanism of great significance, in addition, the epitopes can also be used for the development of peptide vaccines and new drugs. Phage display technology is an inexpensive and rapid method which can be used to mapthe epitopes. In the late1980s, Stephen developed a phage vector system and recently, the use of the phage vector system prepared by phage random peptide library has been applied to the protein epitope analysis.In this study, PEDV N gene was cloned by RT-PCR, resulting in a recombinant plasmid pET-30a-N. The recombinant plasmid pET-30a-N was transformed into the host expression bacteria Rosseta and expression was induced with a final concentration of1mmol/L IPTG. SDS-PAGEshowed that the recombinant N protein was about60Ku. Four anti-N protein monoclonal antibodies were generated by immunizing the N protein into BALB/c mice followed by hybridoma techniques.Using phage random peptide library,4panning,12positive phage clones recognizing a monoclonal antibody3F8were achieved. Sequencing results showed that11phage clones had consensus sequences TPXXWXYXXXXGP, which shared similarity with PEDV N protein amino acid70-82(QPSNWHFYYLGTGP) in PEDV N protein. A phage clone sequence DPGHRLLAWDRL displaying HXXLXWDXX(X represents the random amino acid residues) had a homology with amino acid414-422(HANLEWD) in the N protein. Phage-based ELISA and antigen competitive inhibition test confirmed that the phage displayed epitopes were similar with the natural epitopes in the PEDV N protein, therefore, they can be used to detect PEDV antibody and can also be used to develop epitope vaccines. This study is helpful for PEDV diagnosis and prevention.