Establishment Of Indirect ELISA For Detecting Method And Preparation Of Monoclonal Antibody Of PEDV S Recombinant Protein

Porcine epidemic diarrhea is a highly contagious disease which caused by porcineepidemic diarrhea virus, vomiting and severe diarrhoea dehydration as the main clinicalfeatures. Different age and different varieties of pigs are susceptible, lactation pigletmortality is highest. The mortality rate is more than95%. In recent years, the popular areaof porcine epidemic diarrhea is gradually expanding trend, more serious harm, bringimportant economic losses to the pig industry. Therefore, timely diagnosis and preventionof PED’s of great significance.According to the S gene sequence of porcine epidemic diarrhea virus classic strain inGenBank, then refer to PMD-19T vector map,1pairs of primers were designed whichwere inserted into the restriction endonuclease BamHⅠand XhoⅠsites, Using polymerasechain reaction, a1080bp fragment in suspected of porcine epidemic diarrhea diseaseHB/FN strain was obtained and connected into PMD19-T vectors, and then transformedinto competent cells of Escherichia coli DH5α.The recombinant plasmid was indentifiedby enzyme digestion and DNA sequencing. The sequencing results showed thatits homology with S from GenBank was not less then94%which indicated that theconservatism of S gene was high in different isolated srrains.Based on the antigenicity analysis of S gene, a pair of specific primer was desigined.Afragment of higher antigenicity at lengeh of655bp was amplified by polymerase chainreaction. The fragment was inserted into pET-28a(+) to construct prokaryotic expressionvector. The recombinant expression plasmid pET-28a(+)-SFN was transformed intocompetent cells of DE3and induced by IPTG, SDS-PAGE analysis showed that S genecould be expressed the vector with high expression level,the product is about25ku, andthe same of the expected results. After purification, Western-Blot assay indicated thatthe target protein could react with PEDV immune serum which shows good antigenicity.Using the purified fusion protein as coating antigen, after several experimentsanalysis, the method for detection is finalized: Coating antigen4℃overnight, closed1h in blocking buffer37℃,adding100ul serum to be tested (diluted1:100in blocking buffer)reaction at37℃for1h, adding100ul secondary antibody (diluted1:1000in blockingbuffer) at37℃for45min, added100μL TMB, keep it away from the light for15min;measured after adding the Stop Solution50μL of OD450nm. The method does not cross-reactwith positive serum of TGEV, CSFV, PRV, PRRSV. The repeatability test results show thatthe mean coefficient of variation of less than10%, indicating that the method has goodspecificity and reproducibility.Then use the PEDV purified recombinant S protein as an immunogen to immuneBALB/c mice by a conventional method.Using, Prepare monoclonal antibody (McAb) bythe hybridoma technique.After cell fusion,the use of lymphocyte hybridoma technologyPEDV strains of anti-monoclonal antibody was prepared, and named1F2.ELISA resultsshowed that the monoclonal antibody and S protein antigen was positive. Western-blotindicated that IF2had special reaction with the S protein of PEDV,can be further used inthe preparation of colloidal gold immunochromatographic strip.