Construction Of PEDV Vaccine Transfer Vector And Rescue Of Recombinant Virus

Porcine epidemic diarrhea (PED) is a highly contacting transmitted disease that ischaracterized by acute enteritis, vomit, diarrhea and dehydration. The causative agent of PED isPorcine epidemic diarrhea virus (PEDV). In recent years, the disease widely spread in China,resulting in serious economic losses, attracting much attention of various Chinese academicinstitutions.Currently, protective efficacy of PEDV commercialization vaccine is reduced year by year.There are two main reasons. First, the antigenicity of current commercialization vaccine does notmatch with the epidemic PEDV strains. Second, deactivation of vaccine strains being excessivelyweakened since in vitro passage culture and adaptation. Therefore, the development of a newgeneration of vaccines is of momentous significance. This study was designed to get recombinantvirus through reverse genetics system of PEDV based on RNA targeted recombination by themethod of S gene replacement for providing PEDV vaccine candidate strains.The study is divided into two parts.Part1: Construction of PEDV vaccine transfer vector. Select three epidemic PEDVcell-adapted strains S genes (KF804028, KF650372, KF650375) as immunogenic genes. on thebasis of the S gene sequence of three prevalence strains （genbank： KF804028, KF650372,KF650375），we synthesized the primers into small fragments by overlap PCR. Then smallfragments are ligated to pJET-Blunt cloning vector. The thirteen small fragments with correctsequence are amplified by PCR. The adjacent small fragments which have21bp overlap regionfuse to form short segments by fusion PCR. As the same method,3S genes of epidemic PEDVstrains are synthesized. Three S genes of epidemic PEDV cell-adapted strains are ligated top-PEDV-ΔS DR13by homologous recombination. The correct recombinant plasmids are finallyobtained by sequencing. The corresponding recombinant plasmids were designated pPEDV-DR13ΔS/KF804028S、pPEDV-DR13ΔS/KF650372S、pPEDV-DR13ΔS/KF650375S. Part2: Rescue of recombinant virus. Three recombinant plasmids carrying different S genesof epidemic PEDV cell-adapted strains are transcribed into RNA in vitro. By electroporation, Sgenes of epidemic PEDV cell-adapted strains are integrated into the viral genome. Following that,we infect vero cells with rPEDV. Finally, rPEDV with the ability to produce CPE on vero cells andserially passage is rescued. The corresponding recombinant viruses were designated PEDV-DR13ΔS/KF804028S、PEDV-DR13ΔS/KF650372S、PEDV-DR13ΔS/KF650375S.