Expression Of Porcine Epidemic Diarrhea Virus S1 Protein In Mammalian Cells And The Establishment Of An Indirect ELISA

Porcine epidemic diarrhea is a highly infectious disease caused by porcine epidemic diarrhea virus.It is characterized by severe diarrhea,vomiting and dehydration.The morbidity and mortality of newborn piglets are extremely high after infection.Since 2010,The G2 strain has caused a large-scale outbreak of PED in China.Immunization is an impo rtant means to prevent and control PED,however,there is a lack of effective methods for PED immune surveillance at present,and it is impossible to evaluate the antibody level in pigs infected or immunized.Therefore,the purpose of this study is to esta blish an indirect ELISA which can be used for PED immune evaluation.The main research contents are as follows:1.In order to establish a stable ST cell pool expressing PEDV S1 protein,we used EGFP as a reporter gene to explore the best electroporation trans fection conditions and G418 pressurized screening concentration of ST cells.Finally,the best electrotransfection conditions of ST cells in 4mm cup were determined as follows: plasmid dosage was 20 μg,voltage was 380 V,pulse time was 40μs,electric shock once,and the best pressurized screening concentration of ST cell G418 was 200 μg/m L.2.The PEDV S1 gene was cloned into the p IRES1-neo mammalian cell expression vector to construct the p IRES-S1 recombinant expression plasmid.The plasmid p IRES-S1 was transfected into ST cells by electrotransfer.After G418 pressurization screening,Western blot detection and suspension domestication,a stable transduction cell pool expressing S1 protein was obtained.The molecular weight of PEDV S1 protein obtained in thi s study is about 120 k Da,indicating that the PEDV S1 protein expressed in ST cells has been modified by glycosylation.The results of western blot showed that PEDV S1 protein reacted strongly with PEDV positive serum,indicating that it had good reactivity.3.Using PEDV S1 protein as coating antigen,an indirect ELISA was established,and the conditions were optimized,such as the coating concentration of antigen,the dilution ratio of serum and HRP conjugate,the action time of TMB substrate.This indrect E LISA has no cross reaction with CSFV,PCV2,PRV-g E,PRV-g B,PRRSV and FMDV positive sera.The indrect ELISA can detect PEDV positive serum which diluted 6400 times.The coefficient of variation of intra-assay and inter-assay repeatability is less than5%.the stability of the antigen plate is good,the overall average coefficient of variation between the antigen plates stored at room temperature(25 ℃)for 6days is 4.25%.Taking the serum neutralization test as the standard,the sensitivity of the indrect ELISA was 96.3% and the specificity was 97.7%,which was highly consistent with the serum neutralization test(Kappa value =0.882,P < 0.05).Compared with the IDEXX PEDV Ig A antibody detection kit,the results showed that the S1-ELISA had a higher detection rate of antibodies in the serum with lower neutralization titer,which could more accurately reflect the level of anti-PEDV neutralizing antibodies in pigs.4.The established S1-ELISA was used to detect the clinical serum of PEDV.The results showed that the S1-ELISA could be used to detect the level of specific anti-PEDV Ig G antibody in infected or immunized pigs,and the antibody level is related to the clinical protection status,which is of great significance to evaluate the immune protection status o f Porcine epidemic diarrhea in pigs.