Screening And Characterization Of Chicken Amino Peptidase N Protenin Affinity Peptides

Viral receptor is a molecular complexes that present in the host cell surface, can be recognized and combined by adsorptive protein of virus. Aminopeptidase N (APN) is a zinc-dependent metallic glycoproteins locating on the cell surface and is a cell receptor of most of type â…  and type â…¡ coronavirus. Infectious bronchitis virus (IBV) is one of avian coronavirus belonging to type â…¢ coronavirus.It is considered to be coronavirus between type1and type II coronavirus evolutionarily. Currently, functional cell receptors of IBV has not been completely determined, chicken aminopeptidase N is most widely researched in the study of IBV functional cell receptor. More detailed indepth exploration whether chicken APN is the function receptor of IBV is very valuable.In this study, total RNA was extracted from kidney of18-day-old chicken. With RT-PCR and SOE PCR technique, the gAPN gene was cloned in3parts and linked together of totally2906bp of entire open reading frame. The gAPN was expressed after construction of pET-gAPN and transformed E. coli Rosetta, the molecular weight of gAPN recombinant protein is113kDa. The purified gAPN recombinant protein was used as antigen for preparation of rabbit anti-gAPN polyclonal antibody, indirect ELISA showed that polyclonal antibody titer was up to215, Western blot showed that this polyclonal antibody had highly reactivity and specialty with gAPN recombinant protein and natural gAPN from chicken kidney. To further test antiserum activity, we transfect eukaryotic plasmids pcDNA-gAPN into Hela cells, and specific binding signal was detected. This indicates that polyclonal antibody has good biological activity.Using phage display technology, prokaryotic recombinant protein of gAPN were panninged with Random12Phage Display Peptide Library, After four rounds of screening randomly, high affinity phages were selected and sequenced, and2kinds of afffnity peptides with peptide sequences of HDYLYYTFTGNP(H) and TKFSPPSFWYLH(T) were synthesized. Viral suppression experiments in vitro showed that peptide H and T could inhibit IBV replication in chicken kidney cells. The antiviral experiments of peptide H and T in vivo show that affinity ploypeptide H, T immunized chicken could produce specific antibody, which could react with IBV S protein and could inhibit the reproduction of the virus in chickens traechea,lung and kidney. The results of this research provide the experimental materials for the further examination whether APN can be used as IBV infection functional receptors and some theoretical and research basis for development of related peptide vaccine.