Expression And Identification Of Pig Aminopeptidase In MDCK Cells

It was reported that aminopeptidase N (APN) is the receptor for Coronavirus.The Class I Coronavirus which contains Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus(PEDV), Human coronavirus 229E (HCoV-229E) and Feline coronavirus (FeCoV) utilized tissue-infectious receptor as aminopeptidase.The recombinant pEGFP-C1-pAPN plasmid was constructed by insertion of gene pAPN into eukaryotic expressing vector pEFP-C1 with BamHâ… a nd Hindâ…¢digestion sites. According to sequencing results, the gene pAPN have correct DNA sequence. After analysis of 3D-constructure predicted software, the pAPN was able to fused into GFP to express without any change and infulence between them corresponding to natural structure. The recombinant plasmid was transfected into MDCK (Madin-Darby canine kidney) cell non-available for PEDV via liposome media, Real-time detection of GFP expression was performed via fluorescence and selection by addition of neomycin G418 into tissue culture because of resistant gene involved in vector pEFP-C1. Several MDCK cell lines wiht G418 resistance were selected, at this same time, the total RNA of cell infected by PEDV was isolated to reverse transcription with OligoT, a pair of primers pointed to pAPN was designed to detect three MDCK cell lines by RT-PCR.Three MDCK cell lines were infected with PEDV but there were two strains with CPE, which demonstrated the expression of pAPN in the cell.The kind of MDCK cell was passaged 10, after deletion the pressure of G418, 20 passages were cultured sucssivly, the obvious CPE were able to be found after infection by PEDV,which demonstrated the stable expression of pAPN in this MDCK cell. After verification of anti-S protein and anti-pAPN serum neutralization test, furtherly certified that pAPN played an important part during infectious course, which presumed S involved in this stage, it means that the receptor was pAPN and the ligand was S. Two strains of MDCK cell lines were obtained in this experiment, which reflected PEDV propagation in the cell.The theoretical and practical systems were established to be helpful to research in the function of coronavirus receptor.