Screening And Characterization Of M Protein Affinity Peptides Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED) is acute, highly contagious gastrointestinal tract diseases caused by porcine epidemic diarrhea virus(PEDV). The clinical feature is acute vomiting and severe diarrhea, and can lead to high mortality in neonatal piglets. PED was reported outbreak frequently worldwide and resulted in serious economic losses in swine industry. The M protein is the major structural protein of PEDV, with highly conservation, is one of the most important proteins to stimulate host immune protection. It decides virion assembly sites, virus maturation and budding sites. In addition, specific M protein antibody in the presence of complement can neutralize virus infection. Therefore, the study of the biological activities of PEDV M protein has great significance in illustrating the infection mechanism of PEDV and comprehensive control of this disease.In this study, PEDV M protein positive plasmid pET-32a-PEDV-M was constructed and expressed in E. coli expression system. Recombinant M proteins of 41 kμ was got and in the form of inclusion body. The purified protein was used as antigen to immunize the white rabbit, and then, polyclonal antibody was prepared, and its biological activity was characterized through the indirect ELISA or Western Blot or IFA assay. The results showed that: the titer of polyclonal antibody was approximately 105, moreover, the polyclonal antibody, which has good antigen-antibody reactivity with PEDV.The purified and renaturated M protein was used as target protein and screened with phage display random 12-mer peptide library for 4 rounds, 10 phage clones with high affinity of PEDV and recombinant M protein were obtained, sequenced, and the deduced amino acid sequence were as followed: AGWYCTEVLCV, AYCTRHVCYLDN, which designed M-2, M-9, respectively. The antiviral activity of these two synthesized polypeptides was evaluated in vitro. Indirect fluorescence assay(IFA) and real-time quantity reverse transcription polymerase chain reaction(q PCR) showed that M-2, M-9 polypeptide alone or combined could effectively suppress viral replication, and in a dose-dependent manner. The antiviral activity of M-2 polypeptide was better compared with that of M-9 polypeptide, and it had the highest inhibition on PEDV multiplication of M-2 and M-9 polypeptide combined treatment. In summary, it provided some reference to identify and characterize the functional ligands PEDV M protein, as well as provided theoretical and experimental basis for the development of small therapeutic molecules for PED prevention in the future.