Enfeksiyöz Bovine Rhinotracheitis Virusun Tespitinde IBRV Suş SD Rekombinant Glikoprotein D Kullanılarak İndirekt ELISA Yönteminin Geliştirilmesi ve Uygulanması

Glycoprotein D (gD) is the major structural protein of infectious bovine rhinotracheitis virus (IBRV). It can induce both humoral and cellular immunity thus it is a preferable protein for IBR diagnostic reagent. Regarding to DNAstar analysis, the major antigenic region of the gD fragment was amplified by PCR using the IBRV genomic DNA as a template, and subsequenly constructed into the recombinant plasmid pET32a-gD. The fusion protein was expressed upon IPTG induction. The fusion protein was purified by immobilized Ni ion affinity chromatography with a Ni-NTA Kit after verification by SDS-PAGE and Western-blotting analysis, then was utilized as a coating antigen to detect antibodies to infectious bovine rhinotracheitis virus (IBRV) in an indirect ELISA method. Cross-reactivity examinations have showed that the recombinant antigen had no cross reaction with positive sera of other common viral diseases (bovine ephemeral fever, bovine viral diarrhea-mucosal disease, calf diarrhea, bovine intestinal virus infection, bovine coronavirus disease) which indicating a strong specificity. Application of this diagnostic method in 1315 clinical serum samples displayed an antibody positive rate of 23.7% (311/1315), with respect to a coincidence rate of 96.8% as compared to a commercialized IBRV whole virus ELISA. Our method is stable and sensitive hence provides a quick and convenient serological diagnosis favoring epidemiology and disease identification of domestic IBR.