Expression Of Bovine Infectious Rhinotracheitis Virus GD Gene And Establishment Of ELISA Diagnostic Method

Infectious bovine rhinotracheitis virus (IBRV) is main pathogen of Infectious bovinerhinotracheitis virus (IBR). IBRV often caused severe respiratory and genital tract infection ofbovine. Glycoprotein gD protein was located on the surface of virus envelope, which canstimulate organism to generate neutralizing antibody, so it could be used to diagnostic reagent forIBRV. In the present study, prokaryotic expression of IBRV gD gene to establish an indirectELISA method to detect antibodies against IBRV, expecting that it can fast diagnose IBR.Thisstudy mainly contains two researches:A pair of primers was designed according to the IBRV gD sequences in GenBank to amplifythe complete open reading frame. PCR product of gD, containing three epitope: gD-A、gD-B、gD-C, was then inserted into into the prokaryotic expression vector pET-28a-gD respectively,then transform the recombinant plasmid into competent cells of BL21(DE3). After induced byIPTG, both of them were successfully expressed and identified by restriction enzyme digestionand gene sequencing. SDS-PAGE demonstrated that gD fusion protein was effectively expressedas inclusion body in Escherichia coli about48ku. Their molecular weight accord with expection.The concentration of the purified recombinant protein was0.128mg/ml for gD. Western blotproved that the purified gD fusion protein can be identified by the immunized IBR positive sera,demonstrating a good immunogenicity.An indirect ELISA detection method was successfully established using the purifiedrecombinant gD protein as antigen, and through optimal reaction condition, antigen coated wasat6.4μg/mL，coating antigen overnight at4℃, serum dilution was at1:50, serum reaction1hat37℃, blocking solution at1%skim milk, HRP-rabbit anti-bovine IgG dilution at1:10000,and substrate reaction15min at37℃, the critical value of negative-positive sample was0.219.The method had a high specificity and fine reproducibility andit is relevance ratio90.2%withIDEXX test. The method was used to detect442bovine serum samples from Heilongjiangprovince, with the positive rate of serum antibody23.1%(102/442). This result indicated thatELISA method had a fine sensitivity and specificity, having very broad application prospects.