Preparation Of Polyclonal Antibody Against Recombinant E2 Protein Of BVDV And Establishment And Application Of An Indirect ELISA

Bovine viral diarrhea/mucosal disease (BVD/MD) is caused by infection of cattle with bovine viral diarrhea virus (BVDV). BVDV infection causes great losses in the cattle industry world-wide due to a wide spectrum of clinical and pathological conditions, varying from reproductive failure, congenital abnormalities and general immunodepression to unthrifty calves and severe mucosal disease. The genome of bovine viral diarrhea virus encodes eleven proteins. Among them glycoprotein E2 is one of the major protective antigens of virus. It may induce the production of neutralizing antibody, protecting experiment animals from homologous virus challenge. E. coli BL-21(DE3) was transformed with the recombinant plasmid pET30a-E2. The recombinant E2 protein was expressed in E. coli after cultivation and induction. The purified recombinant E2 protein could be recognized by specific BVDV antisera in Western blotting. Then the purified recombinant E2 protein was used as antigen for immunizing rabbit to obtain polyclonal antibody against the recombinant E2 protein. The result of virus neutralization test showed that the titer of the polyclonal antibody to neutralize BVDV was 1:2048, the polyclonal antibody has a good specificity. The polyclonal antibody against the recombinant E2 protein of BVDV also had highly reactivity and specialty in immunofluorescence analysis and Western blotting. Indirect immunofluorescence test to prove its 1:1280 dilutions still has a higher activity. The polyclonal antibody against recombinant E2 protein of bovine viral diarrhea virus (BVDV) developed in rabbits could be used in detection of BVDV in China and provided a good basis for establishing an ELISA for detecting of E2 protein of BVDV.Using the recombinant and purified E2 protein as antigen, an indirect ELISA was successfully developed to detect antibody to BVDV. The indirect ELISA was named E2-ELISA. The optimal working parameters were determined as follows;the recombinant E2 protein antigen for coating at 12.8μg/ml, testing sera dilution at 1:160, serum reaction time at 60 min , blocking solution at 1% gelatin, blocking solution reaction time at 60 min, anti-bovine IgG peroxidase conjugate dilution at 1:5000, anti-bovine IgG peroxidase conjugate reaction time at 60 min, substrate reaction time at 10 min, the standard of determining as positive sample OD≥0.364 and negative sample OD<0.364. The recombinant E2 protein antigen showed no cross-reaction with the positive sera of the six kinds of bovine diseases. BVDV can block recombinant antigen react with positive serum in a deep degree, however, it has no clear effect with negative serum. Coefficients of variability percent(C.V %) of intro-batch and inter-batch duplicativity test were less than 10 %. Comparing with the virus neutralization testing (VN), the agreement rate was 86.3%, the specificity was 82.6% and the sensitivity was 87.7%. The agreement rate,sensitivity and specificity of BVDV-E2-ELISA relative to BVDV antibody test kit was 87.5%, 85% and 88.3%, respectively. In the field test, BVDV-E2-ELISA was used to detect 852 serum samples collected from Heilongjiang Province, the result of E2-ELISA demonstrated that the average seroprevalence was 84.18% (632/852). Therefore, this BVDV-E2-ELISA based on recombinant E2 protein antigen has good specificity and sensitivity, suggesting an application prospection.In conclusion, BVDV E2 gene was successfully expressed in inclusion body forms in E.coli and purified, then the rabbit polyclonal antibody having highly reactivity and specialty against the recombinant E2 was obtained. The simple and fast diagnosis method using recombinant E2 protein expressed as diagnosis antigen can be a technique support for further quarantine, prevention and control of BVD in China.