| Japanese encephalitis virus(JEV)is a positive single-strand RNA virus that can cause orchitis of boar and abortion of sows.JEV is a zoonosis virus that can be spread to human by mosquito biting pig.The main encoding protein of JEV include capsid C,membrane PrM/M and envelope,and(NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5)7 nonstructural proteins.JEV wild strain is a derivative in the translation of NS2A gene by ribosome frameshift.The derivative fusion with the C terminal of NS1 protein to derive to NS1'With the G to A at 66th nucleotide of NS2A of JEV vaccine strain SA14-14-2,the vaccine strain lost the structural basis of frameshift to produce NS1'.Meanwhile,NS1 protein was secreted into serum as hexamer dimer and exist in serum for 2 weeks.Therefore,NS1' is a characteristic index of JEV field virus infection.In this study,we immunized rabbit with JEV NS1 protein and got the purified NS1 poly antibody.We use NS1 polyclonal antibody as capture antibody and the HRP cogulated JEV NS1' monoclonal antibody as detection antibody to establish the sandwich ELISA.We expressed NS1' protein in vitro as the positive standard and got the relation curve of protein concentration and OD450nm.Based on this method,we detect the concentration of NS1' protein in the field virus infected cell and the vaccine virus infected cell,also the NS1' concentration in the serum of experimental animal model to measure the change of JEV in vivo and in vitro growth.Meanwhile,we conducted epidemiological investigation of porcine serum samples collected by our lab in 2014 to understand the JEV field virus infection status in swine herds in order to provide suggestion for the control and prevention of JEV in practice.Followed the detailed contents:1?Production and identification of JEV NS1 and NS1' antibodiesWith the construed NS1 prokaryotic expression plasmid,we got purified NS1 protein and immunized rabbits three times to get anti JEV NS1 polyclonal antibody.After purificating by Protein G we got purified NS1 antibody.Western blot and IFA results showed that the purified NS1 polyclonal antibody could react with JEV NS1 protein(about 50kDa)specialized.With the recovered murine NS1' protein monoclonal antibody hybridoma cell,we immunized mouse to get the ascites.After purification by Protein Q we got purified NS1' monoclonal antibody.Western blot result showed that the monoclonal antibody can react with NS1' protein(about 52kDa)specialized.IFA result showed that monoclonal antibody had specialized reaction with JEV field virus infected BHK cell,but had no reaction with JEV vaccine infected BHK cell.We labeled this monoclonal antibody with HRP(horseradish peroxidase)kit and found the titer of both polyclonal antibody and monoclonal antibody are more than 1:10000.The development of these two antibodies provide the material for the followed establish of sandwich ELISA.2?Establishment of DAS-ELISA detecting JEV NS1' proteinIn this study,we used JEV NS1 polyclonal antibody as the capture antibody and the HPR labeled NS1' monoclonal antibody as detection antibody to establish a sandwich ELISA to detect JEV NS1' protein.Followed the optimal condition:the ELISA plate was coated by purified JEV NS1 protein as the capture antigen.The concentration of rabbit polyclonal antibody for coating is lOug/ml,closed with the phosphate buffer containing 2%BSA in 4? for 12h.Add 1:50 diluted antigen,1.5 h in 37?.Add HRP labeled NS1'monoclonal antibody with 1:500 diluted,1 h in 37?.TMB chromogenic time is 10min in 37?.Criteria for judging:OD450nm of sample>0.268 is positive,OD450nm of sample?0.238 is negative.Between 0.238 and 0.268 is suspected sample..The detection limits of sandwich ELISA with prokaryotic expressed NS1 protein is 0.29ng/ml.The curve flattens when the antigen reaches 25.12 ng/ml which means that the combining antigen amount reaches saturation.Meanwhile,when the antigen titer remains from 0.29ng/ml to 4.64ng/ml,the linear relationship is basic in a straight line with good correlation.Detection of JEV infected BHK cell lysis supernatant fluid and cells,showed that the NS1' protein level began to rise after 48 h to 96 h after infection and reached the peak at 96 h after infection.Measurement showed that the secretion of NS1' protein outside the cell and inside the cell is basic consistent.Animal experiment in mice showed that the NS1' protein secreted to serum began to rise at 7 days after infection and reached the peak at 10 days,around 14d began to decline with detected level at 21d.3?Investigation on epidemiology of JEV wild virus infection in different ecological regions in China in 2014Epidemiological investigation was conducted with the sandwich ELISA.The serum of swines comes from Guangdong,Hainan,Jiangxi,Jiangsu,Shandong,Shaanxi,Gansu and other seven herds of pigs,piglets,pigs and sows clinical swine.The result of porcine serum collected by the lab in 2014 showed that the JEV field virus infection rate in hot season(from July to September)was 40%in detevtive swine samples,significantly higher than the infection rate from March(23.4%)to May(33.86%).The infection rate in cold January was 0%.Also,there is an obvious geographical distribution of JEV Infection in July to September.The infection rate in Jiangxi(53.53%),Guangdong(39.69%)and Jiangsu(37.54%)is significantly higher than other regions Such as Gansu(18.13%),Hainan(17.27%),For different herds,most of the JEV infection focus on suckling piglets(48.80%)and sows(47.62%).The infection rate of nursery(26.87%)and finisher(24.46%)is relative low.According to the epidemiology survey,the JEV infection has character of seasonal,regional and herds related.These findings provide a good reference for the control and prevention of JEV and the epidemiology survey. |
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