Genetic Variation Analysis And Development Of Indirect ELISA Diagnostic Method For Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is an acute intestinal infectious disease of pigs,its pathogen is porcine epidemic diarrhea virus(PEDV).Porcine epidemic diarrhea has become popular in more than ten provinces of southern China,and presented the epidemic situation of high incidence and high mortality since the second half of 2010.In recent years,there are some differences among the epidemic PEDV strains of different regions in genetic relationship,many epidemic strains are far from the traditional vaccine strain in genetic relationship,which makes it very difficult to prevent and control porcine epidemic diarrhea.Porcine epidemic diarrhea is very similar to some other diarrheal diseases in terms of clinical symptoms and pathological changes,we can not make a definite diagnosis according to these.Therefore,we should establish a fast,accurate diagnostic method,which is easy to promote at the grassroots level.In this research,genetic variation of 9 PEDV strains which were popular in Shanghai in 2014 was analyzed,which provides a scientific basis for grasping the epidemic characteristics of PEDV and the prevention and control of PED in Shanghai.In addition,N protein of PEDV was expressed in prokaryotic expression system,and an indirect ELISA diagnostic method was established with recombinant protein as coating antigen.The study can lay a good theoretical foundation for the development of porcine epidemic diarrhea antibody detection kit.Details are as follows:1.Genetic variation analysis of porcine epidemic diarrhea virus in shanghai in 2014S,M and ORF3 genes of 9 PEDV strains were amplified and sequenced.The sequencing results were analyzed and the phylogenetic trees based on S gene,M gene and ORF3 gene were drawn by sequence analysis software tool.There are two insertions and one deletion of amino acid in the sequences of S protein of the 9 strains compared with the reference strain CV777;there are some point mutations in the COE epitope region and the core sequence SS6 of linear epitope S1D6;it is completely conserved in the core sequence SS2 of linear epitope S1D5 and in linear epitope 2C10.There are 4 point mutations in the amino acid sequences of M protein,and there are 7 point mutations in the amino acid sequences of ORF3 protein.The phylogenetic tree based on S gene shows that the 9 strains in this study have a closer relationship with USA pandemic strains in 2013 and South Korea pandemic strains from 2008 to 2009,while have a distant relationship with vaccine strain CV777.The phylogenetic tree based on M gene shows that the 9 strains have the closest relationship with USA pandemic strains in 2013,Thailand pandemic strains from 2007 to 2008 and South Korea pandemic strains in 2007,while have a distant relationship with vaccine strain CV777.The phylogenetic tree based on ORF3 gene shows that the 9 strains have a closer relationship with USA pandemic strains in 2013 and South Korea pandemic strains in 2007,while have a distant relationship with vaccine strain CV777.2.Prokaryotic expression of PEDV N proteinN gene fragment of a shanghai PEDV strain in 2014 was amplified by RT-PCR,and then cloned into pET30a vector,the recombinant expression plasmid pET30a-N was constructed.Then pET30a-N was transformed into E.coli BL21(DE3)and expressed with induction of IPTG.It was demonstrated that the objevtive protein was expressed in BL21(DE3)and mainly existed in the inclusion bodies by SDS-PAGE electrophoresis analysis.The inclusion bodies were dissolved,purified,renatured,and then identified by Western-blot.The identification result manifested that the recombinant N protein had a nice reactionogenicity.3.Development of indirect ELISA diagnostic method for PEDVThe ELISA was performed with recombinant N protein coated microtiter plates.All the reaction conditions of ELISA were confirmed through a series of optimization tests.92 clinical serum samples were detected with the indirect ELISA diagnostic method in this study and PEDV antibody detection kit produced by BIOVET Company at the same time,the coincidence rate of the two detection methods was 89.1%.It showed that the accurate rate of the ELISA diagnostic method was high.Finally,423 clinical serum samples collected from 15 large-scale pig farms of shanghai in November 2014 were detected with this method,and the PEDV antibody positive rate was 41.8%.