Isolation,Identification And Genomics Research Of PEDV SNJ-P Strain And Establishment Of Indirect ELISA Method For S Protein

Porcine epidemic diarrhea?PED?is an acute infectious disease caused by Porcine epidemic diarrhea virus?PEDV?,which can cause acute diarrhea in pigs and rapidly dehydrate and die,which is harmful to suckling piglets.maximum.Since 2010,PED has exploded on a large scale around the world.Variation of the strain is one of the main reasons for the difficulty of controlling the epidemic.In this study,PK-15 cells were used for virus isolation in the intestinal contents of diarrhea suckling piglets.Cell cultures were identified by cytopathic,RT-PCR and agarose gel diffusion assays,and it was confirmed that a PEDV strain was obtained.Named SNJ-P.The virus can be cultured in a virus culture solution having a final trypsin concentration of4?g/mL,and passed through the fourth generation to produce cytopathic effects.Continued passage to the 7th generation,the cells stably CPE.Passage to the 10th generation,the CPE time is advanced to 12 h after the poisoning.The 15th generation cell virus solution was collected for plaque purification,and the TCID₅₀0 of SNJ-P was calculated to be 1×10^-6.5/mL.Observing the pathological changes of SNJ-P artificially infected suckling piglets and detecting the viral nucleic acid load in various tissues and organs,the pathological sections of the main diseased organs were made.The results showed that except for the intestine,the viral nucleic acid load in the lung and liver was the highest,showing intestinal>lung>liver.A total of 21 primers for amplifying the whole genome sequence of PEDV were designed to align the whole genome sequences of the SNJ-P isolates.Analysis of the genetics and variation of each gene fragment,discussion of the variation may cause functional changes in its products,analysis of the current unsatisfactory reasons for vaccine immunity from the genetic level,and provide theoretical assistance for the development of new vaccines.A gene-wide evolutionary tree was established to analyze the molecular genetic evolution relationship between the strain and the reference strain.The results showed that SNJ-P had the highest homology with Chinese strains ZL29 and CH/HNYF,and the homology was 98.4%and 98.7%.It is far from the Russian strain Blegorod and has a homology of 90.9%.The S gene of the isolated strain was analyzed,and a pair of primers were designed to amplify the antigenic determinant gene region?689-794 aa?with high antigenicity,and the recombinant plasmid pET-32a?+?-SJ was constructed.The recombinant S protein with a size of 35 kD was induced and expressed as an inclusion body.The protein was purified by Ni-Agarose His tag protein purification kit to obtain a recombinant protein with a concentration of 1.145 mg/mL.It was identified by Western-blotting and proved to have good antigenicity.The purified recombinant protein was used as a coating antigen,and each reaction condition was optimized to establish an indirect ELISA method for detecting PEDV serum antibody.The criterion is that OD_450nm?0.324 is positive,and vice versa.The coincidence rate with the commercially available kit was 96.67%.The method has good specificity,high repeatability and low cost.It can be used for clinical PEDV serum antibody detection and PEDV epidemiological monitoring,which is of great significance for the prevention of this disease.