The Construction Of PEDA Bacterial Artificial Chromosome Clones And PAPN Stable Expression Cell Lines

Porcine epidemic diarrhea virus disease is caused by porcine epidemic diarrhea virus(PEDV).The main symptoms are vomiting,dehydration,diarrhea and the atrophy of small intestinal villous.Piglets and fattening pigs are susceptible to PEDV.The infection and mortality rates of the piglets are about 80%-100%,resulting in significant economic losses.PEDV are enveloped,single-stranded,positive-sense RNA viruses,and the size of the genome is about 28kb.Here we describe the engineering of PEDV full-length cDNA clones as bacterial artificial chromosomes(BACs).In this study,the complete genome of porcine epidemic diarrhea virus(PEDV-CHZ)isolated from Jiangsu Changzhou was sequenced and analyzed.The whole genome can be divided into four parts based on the analysis of the genome sequence and the pBeloBAC11 The four segment viral gene sequences of PEDV-CHZ strain were amplified by PCR.The pBAC-PEDV-5'-3' intermediate plasmid is assembled in the BAC under the control of the the cytomegalovirus(CMV)immediate-early promoter and it is flanked at the 3'-end by a 25-bp synthetic poly(A)followed by the sequences of the hepatitis delta,virus(HDV)ribozyme and the bovine growth hormone(BGH)termination and polyadenylation signals to produce synthetic RNAs bearing authentic 5-and 3'-ends of the viral genome.This study laid a foundation for the construction of PEDV infectious clone.Porcine aminopeptidase N(pAPN),type II transmembrane glycoprotein,have been identified as the cell receptor of porcine epidemic diarrhea virus.The gene of pAPN was amplified by RT-PCR,and cloned into the pIRES2-EGFP eukaryotic expression vector.The recombinant expression plasmid pIRES2-EGFP-pAPN was successfully constructed.The cell genomic targeting sequence was screened and inserted into the recombinant plasmid by overlapping PCR.The plasmid PX330 and the successfully constructed plasmid containing the target sequence pIRES2-EGFP-pAPAN-R were transfected into ST/PK cells.Three cell lines were obtained through the screening of G418,green fluorescence and limited dilution.Western blot and indirect immunofluorescence test showed that the pAPN transcribed and expressed stably in the constructed ST/PK cells.Our study successfully constructed the eukaryotic expression vector of pAPN and obtained three stable expression cell lines,which laid a foundation for the stable expression of protein as well as the virus receptor related research.