Isolation Of Porcine Epidemic Diarrhea Virus And Screen Of Its Receptor Protein

Porcine epidemic diarrhea virus(PEDV),is an important pathogenic factor causing epide mic diarrhea in pigs of all ages,causing immeasurable economic losses to the pig industry.On ly relying on vaccine immunization and drug treatment can not effectively prevent the occurre nce and spread of porcine epidemic.Disease resistance breeding is an effective way to solve t his problem.Studies have shown that PEDV infection includes adsorption,invasion,replicati on and release processes.Inhibition of viral invasion is a good antiviral strategy.Therefore,sc reening PEDV receptor proteins and resistance molecular markers,breeding PEDV resistant p ig herds is an effective way to defend against porcine epidemic diarrhea.In this study,the two levels of PEDV virus isolation and recipient protein screening were mainly carried out in ord er to isolate the PEDV resistance function genes and molecular markers.The main findings of the study are as follows:Fisrt,we extracted the RNA what come from the intestinal contents of diarrhea piglets,a nd the pathogen was initially identified as PEDV by RT-PCR.Viral was isolated by seeding V ero·E6 cells,and typical cytopathies(CPE)appeared in Vero·E6 cells by blind transmission to the sixth generation.And as the time of infection increased,the cell lesions gradually became apparent and the lesion area gradually increased.Indirect immunofluorescence technique were performed on Vero·E6 cells infected with the virus using PEDV polyclonal antibodies,which further confirmed that we successfully isolated the PEDV strains.We determinated the virus'i nfection amount of half of the cell cultures is 10^-6/m L.The strain that isolated it was named S DTA2021.Flow cytometry assays revealed that the SDTA2021 strain can induce apoptosis by dose-dependenting in Vero·E6 cells.Second,the S,M and N genes of SDTA2021 strains were amplified by RT-PCR by desig n primers.We compared SDTA2021 with 17 representative PEDV strains to carry out genetic evolutionary analysis.The results showed that compared with the classical strain CV777,the S gene was missing ATA at the location of bases 455-457,and there were 122 base mutations.The same as S gene,the M and N genes had 11 and 31 base mutations,respectively.Based on the results of genetic evolutionary analysis,the SDTA2021 isolate obtained in this institute be longs to the same branch as the G1 subtype strain.Compared with the G1 subtype strain,SDT A2021 is relatively close in the affinity and the nucleotide sequence homology is high.While Compared with the G2 subtype strain,SDTA2021 is relatively farther in the affinity and have low nucleotide sequence homology.Bioinformatics analysis based on three genes showed that the SDTA2021 isolate belonged to the G1 strain.The SDTA2021's gene sequence was mutate d to varying degrees,but it was the same as that of the classical strain.Finally,we constructed a yeast double hybrid library of pig small intestine epithelial cells IPEC-J2 by gateway library building technology.Since PEDV invades cells by binding to hy bridization system to screen the S1 protein receptor proteins.We used the vectur to carry out inspection of the self-activating activity and toxicity which was constructed by PEDV S1 prot ein and p DHB1.It was found that the S1 protein has no self-activation phenomenon and no to xic effect on yeast,it is indicated that S1 protein can be used for yeast double hybrid screenin g.And then a positive monoclonal was obtained by converting the bait vector and the library plasmid in the yeast.We selected the single clone to carry out the bacterial liquid PCR,and th e gene was found to be the BCAP31 protein located on the pig X chromosome after homologo us sequence comparison analysis.Studies have shown that the BCAP31 protein is involved in various processes such as promoting the virus to escape from endoplasmic reticulum stress to support the life cycle during viral infection,prompting that the BCAP31 protein may be invol ved in the PEDV virus invasion in the same way.In summary,PEDV was isolated from the intestinal contents of diarrhea piglets in this st udy,and a possible receptor protein BCAP31 was identified by yeast double hybridization tec hnology.This will provide a new research direction for breeding pig breeds with PEDV resist ance from the genetic nature.