关键词: 牛流行热病毒(BEFV); G蛋白; 原核表达; 蛋白纯化; 免疫胶体金

Abstract: 【Objective】 This study was aimed to establish an immunocolloid gold assay for Bovine ephemeral fever virus (BEFV),and provide a research basis for the diagnosis of bovine epidemic fever (BEF).【Method】 The prokaryotic expression vector pET32a-BEFV-G was constructed and transformed into Escherichia coli BL21 (DE3) receptor cells for expression,and the expression product after successful induction was purified and identified by SDS-PAGE and Western blotting.The purified protein was used as the specific antigen-coated detection line,streptococcal G protein was used as the gold-labeled antigen,which was labeled on a gold-labeled pad after binding with gold nanoparticles,and goat anti-mouse IgG was used as the quality-control line.By optimizing the different reaction conditions,the immuno-colloidal gold assay method was established for the detection of BEFV,and the test strips were examined for their sensitivity,specificity and reproducibility.【Results】 SDS-PAGE results showed that the recombinant BEFV G protein was expressed in the form of inclusion bodies with a molecular mass of about 46 ku at a final concentration of IPTG of 0.75 mmol/L and induced at 37 ℃ for 8 h.Western blotting result showed that the concentrated and purified recombinant BEFV G protein had good reactivity and could be used as an encapsulated antigen for the detection of BEFV.It could be used as an encapsulated antigen for the establishment of immunocolloid gold test strips.Under the conditions that the concentration of the antibody sheep anti-mouse IgG encapsulated in the quality control line was 2 mg/mL,the concentration of the BEFV G protein encapsulated was 0.6 mg/mL,and the amount of gold sprayed was 2.5 μL/cm,the detection could be completed in 3-5 min.The prepared test strip had high sensitivity and specificity,and the coloration was close to the elimination line when the serum was diluted 40 times,and the test strip only reacted with the BEFV positive serum,and did not cross-react with the positive sera of other viruses,such as Bovine coronavirus (BCoV),Bovine rotavirus (BRV),and Bovine infectious rhinotracheitis virus (IBRV),etc.The test strips had been proved to be more reproducible after repeated tests of several positive serum samples.The reproducibility of the test strips was proved to be high,and the immunocolloid gold detection method was successfully established.【Conclusion】 In this study,a new type of immunocolloid gold test strip was successfully prepared to detect BEFV using prokaryotic G protein as antigen,which had high specificity and good stability,and was suitable for the rapid detection of BEF in the clinic.

Key words: Bovine epidemic fever virus (BEFV); G protein; prokaryotic expression; protein purification; immunocolloid gold