Isolation Of Procine Epidemic Diarrhea Virus And Prokaryotic Expression Of M Protein

The porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus with symptoms of diarrhea,vomit,dehydration and high lethality rate of piglets, has led to serious economic loss to the world swine industry.From the beginning of winter of2011, piglet deaths caused by the porcine epidemic diarrhea have been observed in most area of Sichuan Province.After the ineffectiveness of antibiotic treatment,we collected the diseased sample and did some laboratory diagnosis.The porcine epidemic diarrhea virus was then found to be the cause of the disease.This work first isolated the PEDV from the sample collected from some large and medium-sized pig farms in Suining area and performed the clone sequence analysis,prokaryotic expression vector construction,and prokaryotic expression to the M Gene.Then we prepared the polyclonal antibody identification to assay the antigenicity,performed the RT-PCR assay to the similar case in Aba and Leshan area,cloned and sequenced the active samples of the RT-PCR assay and did the sequence analysis to the mutated genes.1. The isolation and identification of the PEDVOur experiment successfully isolated the virus using PK-15continuous cell line.In the isolation process,we added the pancreatin with concentration of50um/mL and found that the cells became lighter and turned into a ball.Slight net lesion was observed after the adaption.After the RT-PCR assay,agar diffusion test and animal regression test,the purified virus was identified to be the PEDV virus and named PEDV-Sichuan SC-405,with a TCID50(Tissue culture infective dose) of.2. Cloning and sequence analysis of M gene from the Porcine epidemic diarrhea virusWith the sequencing of the M gene of SC-405, we found the homeology of virus’s nucleotide with that of standard PEDV is97.8%and the homeology of Amino Acid is97.3%. At the same time, we also compared the samples from other areas of Sichuan with the CV777standard strain in the Genbank, and found the nucleotide homeology. From the point of view of the Evolutionary Tree of Phylogenetic, the relationships of the Amino Acid of gene sequence between SC-405M and AFZ45986(Aba, Sichuan), ACA81419(Bangkok NIAH100542-08, Thailand) and BAA25380(Tokyo JMe2, Japan) are close. By the combined analysis of turning angle, random coil, flexible zone, Surface accessible and hydrophilia zone, the feature of intramembranous zone and B-Cell Epitopes to the protein of SC-405M’s gene sequence, we speculate that this protein’s B-Cell Epitopes is most likely distributed at segments Leu15-Asn17, Trp103-Thrl16, Va1199-Asp205, Asp218-Glu220.3. Prokaryotic expression,antibody preparation and antigenic detection of M geneWe cloned the gene of SC-405M and connected it to the plasmid pET-28(a) of prokaryotic expression vector and got the recombination plasmid pET-PM, which was later transformed into the expression host cell BL21(DE3). After many attempts, we found the expression of the inductive object protein of the host cell achieved its maximum after5hours in the IPTG with concentration0.4mmol/L at30degree of Celsius. We observed the reactogenicity of the purified protein after the Western Blot test with the whole virus immune serum of rabbit. Combined with Freund’s adjuvant, the protein can equip the rabbit with polyclonal antibody and immunity. Meanwhile, the protein was shown to have immunogenicity by the agar diffusion test. By all the results, we can see that the prokaryotic expressed PEDV M protein does possess antigenicity.