Prokaryotic Expression Of The Variation Of Porcine Epidemic Diarrhea Virus' N Gene And Development Of Indirect ELISA

Porcine Epidemic Diarrhea(PED)is a kind of porcine digestive tract disease,which caused by Porcine Epidemic Diarrhea Virus(PEDV).PED mainly happens in suckling piglets,causes diarrhea,emesis and dehydration,mortality rate of infected piglets is almost 100%.PEDV belongs to Coronavirus,which possess a single-strand positive sense RNA genome.Genome of PEDV is tend to mutation,which not possible to predict the virology.PEDV was first reported in 1971,and continuous occurs in some countries in Europe and Asia.There is no widely outbreaks of PEDV until 2010,the nationwide outbreak of PEDV varient in China pig farms,which caused large lose in pig industry.In 2013,PEDV infection had been reported in the US,and fast spread to the whole country,even to many other American countries,such as Canada,Mexico and Columbia.In late 2014 and early 2015,PEDV infection had been reported in Europe,the worldwide outbreak of PEDV is still spread.PEDV not only causes damage in pig industry,but also a potential pathogeny to human.Recent studies showed that the mutations in PEDV genome,such as Spike gene,may change the host of virus,maybe even infect human.The outbreak of Severe Acute Respiratory Syndromes(SARS)in China,2003,also caused by Coronavirus.SARS is a nightmare in Chinese public health history,we should do our best to prevent the potential Coronavirus pandemic.The early diagnose of virus infection plays a important role in disease control.The mainly clinic diagnose method of PEDV is RT-PCR,but this method's performing cost a lot of money and time,and relay on PCR thermal cycler.Pig farms and primary level veterinarians can not use RT-PCR to determine disease agent.Enzyme linked immunosorbent assay(ELISA)is a fast diagnose method,and easy to perform.Indirect ELISA established based on recombination PEDV antigen,is an useful tool to evaluate the antibody level in pig serum,which is important in vaccine effect analysis.The IgA antibody in sow colostrum is a important role in piglets's innate immunity,and indirect ELISA could also being use in IgA test,which is guiding data in piglets immune analysis.Nucleocapsid is an structure protein and important antigen of PEDV,nucleocapsid protein(N protein)were high level expressed in infection early stage,and N protein antibody could be detected.PEDV N protein is an ideal antigen to develop indirect ELISA.Thus,two parts of studies have been took in this research.1.The N gene of recent pandemic PEDV variations in China have been sequenced and analyzed.Based on the result,AH2012 strain is the most representative of mutated PEDV.Protein prokaryotic expression vector has been build based on AH2012 strain N gene.And recombination N protein has been expressed,and the immunogenicity has been confirmed positive by Western Blot.Recombination N protein is soluble expression in low temperature,and easy to large-scale preparation and purification in lab,which provided favorable resource to develop ELISA method based on N protein.2.Indirect ELISA method has been established to detect IgG antibody,and each steps of method has been optimized.This indirect ELISA is a stable,accurate method,and rarely influenced by environment factors,such as temperature.Detected results of ELISA is sensitive and specific,there is no cross-reaction to other swine disease positive serum.Results shows,the coincidence rate was 96.3%between this ELISA test and virus neutralization test.PEDV vaccine was used to piglets administration,serum of piglets were collected,and tested by the ELISA.Result shows,serum IgG antibody titer has reached 1:6400 in 1 mouth after the second administration,and the changing rule of antibody is according to nature immune.Based on the indirect ELISA method,an ELISA kit has been developed.The clinic serum testing results showed that,the coincidence rate between ELISA kit and virus neutralization test were 90.77%of positive samples and 80.0%of negative samples.And this indirect ELISA kit could preserve at least 9 mouths in 2?8 ?.This PEDV N indirect ELISA method could application to diagnose,surveillance and antibody level test of PEDV variations in China.And the further develop of PEDV N IgA test indirect ELISA is meaningful in colostrum immune studies.This ELISA method is an practical tool for the prevention and control of PEDV.