Establishment Of An Indirect ELISA Method For Bovine Viral Diarrhea Virus Antibody

This study aim to establish an indirect ELISA method for detecting bovine viral diarrhea virus(BVDV)antibody.BVDV E2 gene sequence published in GenBank was analyzed,by the bioinformatics analysis,using software DNAstar to predict antigenic and hydrophilic domains.The codon was optimized,and the tandem signal peptide sequence was sent to company to synthesize and connect to the prokaryotic expression vector pET-32a(+)to construct the recombinant plasmid pET-32a-Opti-E2.This plasmid was identified transformed into the E.coli BL21(DE3)strain.Expression of size of 43 kDa soluble E2 protein was induced by IPTG,and analyzed by SDS-PAGE and Western Blot.Using soluble E2 protein as coating antigen,an indirect ELISA method for detecting BVDV antibody was established and optimized,named to Opti-E2-ELISA.The S/P values of 203 serum were analyzed by receiver operating characteristic curve method.The negative positive threshold value of S/P was 0.21 in this method.When the S/P value was 0.21,the sensitivity of this method is 96.97%and the specificity is 97.65%.This Opti-E2-ELISA method was also utilized to detect bovine infectious rhinotracheitis virus positive serum,bovine foot and mouth disease virus positive serum,Brucella positive serum and Mycobacterium paratuberculosis positive serum,these results were negative,indicating that the method is specific.Parallel and repetitive tests showed that a coefficient of variation was less than 10%,indicating that established Opti-E2-ELISA method is significantly sensitive,specific and reproducible.Compared to the commercial BVDV antibody detection IDEXX kit,the coincidence rate was 97.5%.