Lingling SONG, Hui ZHANG, Peili HOU, Hongmei WANG, Guimin ZHAO, Xianzhu XIA, Hongbin HE

Development and Preliminary Application of an Indirect ELISA to Detect Infectious Bovine Rhinotracheitis Virus Using Recombinant Glycoprotein D of IBRV Strain SD [

Glikoprotein D (gD) sığır enfeksiyöz rhinotracheitis virus (IBRV)'ün majör yapısal proteinidir. Glikoprotein D hem humoral hem de hücresel bağışıklığı uyarması nedeniyle IBR'nin tespitinde tercih edilen bir proteindir. DNAstar analizi ile ilgili olarak gD fragmanının majör antijenik bölgesi BRV genomik DNA'sı şablon olarak kullanılmak suretiyle PCR ile amplifiye edildi; sonrasında rekombinant plazmid pET32a-gD içinde yapılandırıldı. Füzyon proteini IPTG oluşturulmak suretiyle ifade edildi. Füzyon proteini; SDS-PAGE ve Western Blotting analizleriyle onaylandıktan sonra Ni-NTA Kit kullanılarak immobilize Ni iyon affinite kromotografi ile saflaştırıldı. Sonrasında, saflaştırılan protein indirekt ELISA metodunda enfeksiyöz bovine rhinotracheitis virüs (IBRV) antikorlarını belirlemek amacıyla örtü antijeni olarak kullanıldı. Çapraz reaksiyon incelemeleri ise diğer yaygın viral hastalıkların (bovine ephemeral fever, bovine viral diare-mukozal hastalık, buzağı dizanterisi, bovine intestinal virüs enfeksiyonu, bovine coronavirus hastalığı) pozitif serumları ile çapraz reaksiyon oluşturmayarak güçlü bir spesifiteye sahip olduğunu gösterdi. Tanı amaçlı olarak metodun 1315 serum örneğinde uygulanması neticesinde antikor pozitif oranı %23.7 (311/1315) olarak belirlendi. Bu sonuçla güvenli kullanım oranı ticari IBRV tüm virüs ELISA ile karşılaştırıldığında %96.8 olarak tespit edildi. Çabuk ve kullanışlı serolojik tanı sağlaması nedeniyle mevcut yöntem IBR'nin hastalık tanısı ve epidemiyolojisinde istikrarlı ve duyarlı bir test olarak kullanılabilir.

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.

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