The Sequence Analysis Of S Gene Of Porcine Epidemic Diarrhea Virus Isolated From Anhui And Establishment Of An Indirect ELISA For Detection Of Antibody

Porcine epidemic diarrhea is a highly contagious disease.This disease was first reported in 1971 in the Englands,China's also confirmed the presence of the disease in 1976.The disease has become one of the most serious effects of the global pig industry viral infection,to the pig industry caused significant economic losses.In recent years,China's PED morbidity,mortality showed an upward trend,especially since October 2010,PED widely popular in our country and presented new features.After infection PEDV,piglets mortality rate of 80%-100%,while breeding sows and boars are rarely show clinical symptoms.Mutation of the virus may be the cause of large-scale outbreak of PED,which gives prevention of this disease has brought new challenges.Therefore,for the analysis of genetic PEDV evolution and establish a fast,reliable PEDV antibody detection methods,PED epidemiological surveillance and detection of antibody levels in vaccinated pigs after PED vaccines for the prevention of this disease is important.This study includes the following sections:1: PEDV S Genetic Evolution Analysis Anhui ProvinceWe designed and synthesized three pairs of specific primers amplified S gene by RT-PCR method to investigate the variation in S genes of porcine epidemic diarrhea virus(PEDV)in Anhui during 2014 to 2015.S genes of 21 PEDV strains were cloned and sequenced and their phylogenetic trees were analyzed from different swine breeding farmer.Sequence analysis showed that 96.0% to 99.8% amino acids homologies among 21 PEDV isolates.Compared with CV777 the nucleotide homologies were 93.7% to 95.9% and amino acids homologies were 92.6% to 96.8%.Homology analysis showed that S gene of 21 isolates have insertions and deletions compared with CV777,and these insertions and deletions of nucleotides led to its corresponding changes in encoding amino acids.phylogenetic analysis shoewed that S genes of 21 PEDV strains belonged to the same group except AHBB-1,and had close relationships with 8 China strains isolated in 2011 to 2012 and 2014,three Korea strains in2005 to 2009,,but differ genetically from European strains(CV777,Br1),vaccine strain(CV777-vaccine)and early Chinese strain CH/S.The major S protein antigenic regionof Anhui strains,especially COE domain have amino acids change compared with CV777-vaccine strain.This study would provide the basis for selection and improvement of vaccines and comprehensive prevention and control of PEDV.2:The establishment of an indirect ELISA detection method based on proteinAnalyzing the site antigenic of PEDV S protein by bioinformatics softwares,choose the hydrophilicity strong,antigen index and high surface probability.Design a pair of specific primers for this area,with PEDV genome c DNA as a template for augmentation after reverse transcription,amplification product recycling purification after cloning and prokaryotic expression vectorp ET-32a(+),construct recombinant prokaryotic expression vector p ET-32a-S,the recombinant plasmid transformation induced to the express bacterium BL21,whether with SDS-PAGE protein electrophoresis appraisal expression;By western-blotimmune activity for the identification of recombinant proteins.Grope for the best expression of recombinant protein S conditions,purification of recombinant protein S,with the purified protein S as package material,establish PEDV antibody indirect ELISA detection method,and compared with imported commercialization PEDV antibody kit and Western blot gold standard comparison test,the comparison of the two coincidence rate,sensitivity and specificity.Results show that this study success amplification required S gene and construct the restructuring carrier p ET-32a-S,induce the expressed recombinant protein S,it has been verified by Western blot analysis-good reaction to the original.Indirect ELISA detection method was established through the optimization,the optimal protein was finalized package is concentration of 8 mg/L,sealing fluid is 2% BSA,one in which the best dilution degrees 1:40,best time for 30 min,sheep-HRP best against swine Ig G 1:1500 dilution dilution degree,best time is 45 min,chromogenic time for 10 min.Repeated test showed that coefficient of variation between group and group were less than 10%,good repeatability.Compared with the imported commercial PEDV antibody detection kit coincidence rate,sensitivity and specificity of 86.67%,86.67% and 82.62%;Compared with western blot-test results,the coincidence rate,sensitivity and specificity of 88.89%,90.20%,83.33%.The results showed that the expression of recombinant protein S as envelope antigen to establish indirect ELISA detection method is of high specificity,variability is small,high coincidence rate.Can be used in the production of PEDV antibody levels of detection.