Development Of Double Antibody Sandwich ELISA Detecting Jev NS1' Protein

Japanese encephalitis virus(Japanese encephalitis virus,JEV)is a member of the Flaviviridae and it is the pathogen of Japanese encephalitis,mosquito bites as the main mode of transmission.It is zoonotic disease.Mosquito bites is the main mode of transmission.It mainly caused humans and animals with epidemic brain Inflammation as the main feature of central nervous disease.The clinical manifestations of Japanese encephalitis were high fever,disturbance of consciousness,convulsions,difficulty walking,tonic spasm and other symptoms,some cases of poor prognosis,leaving a serious sequelae.Pregnant sows suffering from the disease will be abortion,stillbirth,mummified tires and other reproductive disorders;boar mainly for the side of the testicular swelling,part of the late atrophy loss of reproductive capacity.The disease has a significant seasonal and a certain regional characteristics,frequent summer.The Japanese encephalitis virus-encoded polyprotein is hydrolyzed to C,prM and E 3 structural proteins and 7 nonstructural protein genes after translation.They can be reverse transcribed,replicated,transcribed and translated into seven nonstructural proteins:NS1,NS2a,NS2b,NS3,NS4a,NS4b,NS5.NS1' protein is a result of the gene transfer code in the process of translation.There are CCCUUUU seven-nucleotide sequence and the secondary stem-loop structure after the sequence in JEV wild-type strain NS2A gene.In the translation process,it prone to shift code,resulting in NS1' protein fusion with the NS1 protein.However,because of gene mutation of the vaccine poison SA14-14-2 NS2A G66A,the stem and loop structure destroyed,which will not produce NSl' protein.So NS1'protein is a unique molecular marker of the Japanese encephalitis virus.In this study,the monoclonal antibody 3C2 of the Japanese encephalitis virus NS1 protein was purified as a capture antibodies of the double antibody sandwich ELISA method.After the purification,the NS1'monoclonal antibody 6F5 of horseradish peroxidase was used as the enzyme-labeled antibody.On this basis,the double antibody sandwich ELISA method was established to detect the Japanese encephalitis virus NS1'protein.Escherichia coli expression of the virus NS1,NS1'protein as the negative and positive standard of the method,then draw the standard curve to determine its detection sensitivity and linear range.The sensitivity and specificity of the method were observed by using the ELISA method to detect the supernatant and the serum of the infected mice.The results were as follows:1.Preparation and identification of NS1' protein and enzyme-labeled NS1'monoclonal antibodyIn order to obtain the NS1'protein of Japanese encephalitis virus,the primers were designed to amplify the encephalized NS1' gene,which was cloned into the pET 28a vector by restriction enzyme digestion.The ligation product was transformed into E.coli DH5a,and the correct positive recombination plasmid was identified by PCR,digestion and sequencing.The plasmid was transformed into E.coli R2 and expressed in a small amount.The expression of recombinant protein was identified by SDS-PAGE.This experiment expressed the NS1' protein which is in inclusion body and is about 50KD.The titer of NS1' monoclonal antibody 6F5 was determined by indirect ELISA.The monoclonal antibody with the highest purity was conjugated with horseradish peroxidase and the titer was tested again.The titer of the monoclonal antibody was 1:204800 and reduced to 1:16000 after coupling.2?Establishment of Double Antibody Sandwich ELISA with Monoclonal Antibody as Capture Antibody and Preparation of Its Standard CurveThe double antibody sandwich ELISA method with monoclonal antibody as the capture antibody was established by square titration method.The optimal reaction conditions were as follows:The purified Japanese encephalitis virus NS1 protein monoclonal antibody 3C2 was used as capture antibody and the coating concentration was 10p.g/mL.Then was blocked with 3%BSA phosphate buffer,37? for 2h;sample was incubated at 37? for 1.5h;HRP labeled Japanese virus NS1' protein monoclonal antibody was diluted in 1:2000,then incubated at 37? for 1h.The limited detection was 0.027?g/mL in a linear range of 0.05?g/mL?0.4?g/mL.The curve tends to be gentle when the concentration of recombinant protein is 10 ?g/mL.The regression equation of standard curve was y= 1.003x + 0.249,r = 0.999,it had a good correlation while the Japanese encephalitis virus NS1 protein can not be detected.The method is specific and sensitive,and is suitable for the Japanese encephalitis wild virus detection in Whether animals or human.3?Detection of BHK cell toxic and animal serum samples by DAS-ELISA(monoclonal antibody)The NS1' protein in BHK cells infected with JEV NJ2008 strain was detected by DAS-ELISA.The OD value was not high.The OD value of untreated or treated at different times with different concentrations of NP-40 and TrisonX-100 was not significantly increased.However,after the heat treatment(serum and DEPC water 1:2 mixed and boiled 5min),the OD value was significantly improved,can reach 0.7 or so.Five mice were inoculated with JEV NJ2008 in 0,102,103,104,105 PFU,and only the serum of 105 PFU was positive with the results of this ELISA.And it is the same with the results of detection of virus nucleic acids in blood and tissue by fluorescence quantitative.The total positive rate was 14.0%for 307 pig serum samples from different provinces using the DAS-ELISA.The positive rate of wild virus infection in September(19.6%)and October(23.3%)was significantly higher than that in February(5.6%).The results showed that the transmission of JEV was obvious seasonal.The results showed that the positive rate of JEV wild virus infection in Zhejiang province(29.7%)and Shandong(38.7%)was significantly higher than that in Jiangsu(9.8%)and Hebei(15.3%)provinces,showing a certain geographical,the positive rate of piglets pig serum higher than litter size piglets and reserve pigs.