The Culture And Isolation Of Wild-type PEDV CH/JX226/2018 And The Research On Anti-PEDV Single-chain Antibody

Porcine epidemic diarrhea(PED)is a kind of acute and highly contagious infectious disease caused by porcine epidemic diarrhea virus(PEDV).The main clinical symptoms are vomiting,diarrhea,dehydration and diarrhea..The high mortality rate of newborn piglets has caused serious economic losses to the pig industry.In recent years,PED has frequently spread out in various parts of the world.Numerous studies have shown that PEDV mutation is the main cause of the outbreak.The variation of epidemic strains does not provide sufficient protection for existing vaccines.Therefore,research on new variant strains is strengthened.It is of great significance to prevent and control PEDV.1.Tissue samples from small intestines of piglets suspected of PEDV infection were collected from a number of pig farms in a certain place in Jiangxi Province.The samples were identified as PEDV-positive samples.After treatment,the cells were isolated and cultured,and morphological and molecular biological identification was performed to maintain The medium containing 2%FBS and 5 ?gg/ml trypsin showed that the cell supernatant contained PEDV when it was blindly passed to the sixth passage,and it was temporarily named as CH/JX226/2018.The S,ORF3,E and M genes of the isolates were amplified and sequenced,and homology was compared with the published reference sequences in GenBank by DNASTAR and MEGA7.0 software to make a genetic evolution tree to analyze PEDV.Sequence characteristics of the major genes of the isolates.2.On the prokaryotic expression vector of pET19b-CBD-scFv against PEDV in the laboratory,using the Fast Site-Directed Mutagenesis Kit,the linker(Gly4Se)3 between the original scFvs was subjected to site-directed mutagenesis,and the linker was obtained.The mutant v1 of Gly4Se,the linker is a mutant v2 of G1ySe,and the mutant v3 without a linker.They were expressed and identified by prokaryotic expression.The renatured proteins were separately bound to the F3 virus solution of the isolated CH/JX226/2018 strain using non-competitive Elisa.To determine the binding strength of scFv and v1,v2 v3.The results showed that the binding force of scFv was significantly greater than v1,v2,and v3.There was no significant difference in binding force between v1,v2 and v3.For the next step of constructing a gene targeting vector,screening for monoclonal cells of the scFv against PEDV provides the gene of interest.3.Using the anti-PEDV scFv as the target gene,designing the gene targeting vector and synthesizing,extracting the plasmid,transfecting with the nuclear transfector,and screening through G418,a total of 86 cloned cells were obtained,and the long arm and short arm wall of the homology arm were passed.Primers were designed inside and outside the wall for PCR identification.The results showed that a primary fetal fibroblast monoclonal cell was successfully screened,and the target target rate of the target gene was only 1.2%,which was the transgenic pig of the future PEDV scFv.Preparation provides cells.