| Infectious bovine rhinotracheitis(IBR) is an acute, feverish and contagious infectious disease of cattle, caused by infectious bovine rhinotracheitis virus (IBRV). IBR is B species contagious disease lined up by World Organisation for Animal Health(OIE), and also focal point quarantine object for country to enter border animals and international animal trades had rated. Accordingly, strengthen IBR diagnostic technique of research work has significant practical importance to prevent and control primary disease. At present domestic don't have commercial detect diagnostic reagent for IBR, seaport detect IBR depend on abroad kit seriously. So this research induce and express IBRV gD gene, using the recombinant and purified gD protein as coating antigen, developed an indirect ELISA to detect antibody of IBRV.Glycoprotein D located at peplos and infect cell surface, it's major structural protein to stimulate organism generate neutralizing antibody, it's specific antigen and leader protein for IBR diagnostic reagent. The recombinant plasmid pET30a-gD was transformed into E. coli BL21 (DE3), the recombinant protein were expressed in partially soluble form after induction with IPTG, and was purified by immobilized Ni ion affinity chromatography under native conditions. The concentration of purified protein was 0.128 mg/ml with high purity (96%). Western blot and indirect enzyme-linked immunosorbent assay analysis suggested that the recombinant protein was recognized by IBRV anti-sera, having a good responsiveness.Using the recombinant and purified gD protein as antigen expressed in Escherichia coli BL21, an indirect ELISA was successfully developed to detect antibody to IBRV, the indirect ELISA was named gD-ELISA, and through a series of test determinate the optimal working parameters, including TMB at 0.4 mg/ml, DMSO at 15 %, 0.75 % urea hydrogen peroxide at 25.6μl/ml, coating antigen at 6.4μg/ml, testing sera dilution at 1:80, serum reaction time at 1.5 h, blocking solution at 10 % horse serum, blocking solution reaction time at 1 h, anti-bovine IgG peroxidase conjugate dilution at 1:5000, anti-bovine IgG peroxidase conjugate reaction time at 1h, substrate reaction time at 15 min, stop buffer reaction time at 5 min, the standard of determining as positive sample PR≥38.0 and negative sample PR<30.8. The recombinant gD protein antigen showed no cross-reaction with the positive sera of the five kinds of bovine diseases. IBRV strain can block recombinant antigen react with positive serum in a deep degree, however, it has no clear effect with negative serum. Coefficient of variability percent(C.V %) of intro-batch and inter-batch duplicativity test was less 5 % and 15 % respectively. Comparing with the Virus Neutralization testing(VN), the agreement rate was 84.1 %, the sensitivity was 85.0 %, the specificity was 83.4 %. The agreement rate, sensitivity and specificity of IBRV gD-ELISA relative to IBRV whole virus ELISA was 91.9%, 94.2 % and 90.2 %, respectively. In the field test, IBRV gD-ELISA was used to detect 2012 serum samples collected from eleven provinces and autonomous regions in China, the result of gD-ELISA demonstrated that the average seroprevalence was 46.0 %(926/2012). Therefore, this IBRV gD-ELISA based on recombinant gD protein antigen has good specificity and sensitivity, suggesting an application prospection.In conclusion, IBRV gD gene was successfully expressed in soluble forms in E.coli and purified, they lay satisfactory foundation for further studies of the protein structure and function of glycoprotein D. The simple and fast diagnosis method using recombinant gD protein expressed as diagnosis antigen can be a technique support for further quarantine, diagnosis, antibody monitoring, prevention and control of IBR in China, and also settle foundation for developing the commercial kit of diagnosis.
|
PDF Full Text Request