Porcine　epidemic　diarrhea　(PED)　has　caused　huge　economic　losses　to　the　global　pork　industry.　Infection　by　its　causative　agent　PED　virus　(PEDV),　an　Alpha-coronavirus,　was　previously　proven　to　be　mediated　by　its　spike　(S)　glycoprotein　and　a　cellular　receptor　porcine　aminopeptidase　N　(pAPN).　Interestingly,　some　recent　studies　have　indicated　that　pAPN　is　not　a　functional　receptor　for　PEDV.　To　date,　there　is　a　lack　of　a　direct　evidence　for　the　interaction　between　pAPN　and　PEDV　S　protein　in　vitro.　Here,　we　prepared　pAPN　ectodomain　and　the　truncated　variants　of　PEDV　S　protein　in　Drosophila　S2　cells.　These　recombinant　proteins　were　homogeneous　after　purification　by　metal-affinity　and　size-exclusion　chromatography.　We　then　assayed　the　purified　target　proteins　through　immunogenicity　tests,　PEDV　binding　interference　assays,　circular　dichroism　(CD)　measurements,　pAPN　activity　assay　and　structural　determination,　demonstrating　that　they　were　biologically　functional.　Finally,　we　characterized　their　interactions　by　gel　filtration　chromatography,　native-polyacrylamide　gel　electrophoresis　(PAGE)　and　surface　plasmon　resonance　(SPR)　analyses.　The　results　showed　that　their　affinities　were　too　low　to　form　complexes,　which　suggest　that　pAPN　may　be　controversial　as　the　genuine　receptor　for　PEDV.　Therefore,　further　research　needs　to　be　carried　out　to　elucidate　the　interaction　between　PEDV　and　its　genuine　receptor.　©　2018