基于牛流行热病毒G蛋白的免疫胶体金检测方法的建立  

Establishment of an Immunocolloidal Gold Assay Based on Bovine Epidemic Fever Virus G Protein

作　　者：王雪妍 靳双媛 杜家伟 温宏武 李永琴[1] 许立华[1] WANG Xueyan;JIN Shuangyuan;DU Jiawei;WEN Hongwu;LI Yongqin;XU Lihua(College of Animal Science and Technology,Ningxia University,Yinchuan 750021,China;Husbandry Service Center of Yanchi Agriculture and Rural Bureau,Wuzhong City,Ningxia Province,Yanchi 751500,China)

机构地区：[1]宁夏大学动物科技学院,银川750021 [2]宁夏吴忠市盐池县农业农村局畜牧技术推广服务中心,盐池751500

出　　处：《中国畜牧兽医》2024年第10期4429-4439,共11页China Animal Husbandry & Veterinary Medicine

基　　金：宁夏奶牛育种专项“优质高产奶牛安全健康养殖技术研究与示范”(2019NYYZ0503);宁夏动物疫病净化科技创新团队项目(2020CXTDLX05)。

摘　　要：【目的】建立牛流行热病毒(Bovine ephemeral fever virus,BEFV)的免疫胶体金检测方法,为牛流行热(BEF)诊断提供研究基础。【方法】构建原核表达载体pET32a-BEFV-G,将其转化大肠杆菌BL21(DE3)感受态细胞进行表达,对诱导成功后的表达产物进行纯化和SDS-PAGE、Western blotting鉴定。以纯化后的蛋白作为特异性抗原包被检测线,以链球菌G蛋白作为金标抗原,与金纳米颗粒结合后标记在金标垫上,用羊抗鼠IgG作为质控线,通过优化不同的反应条件,建立检测BEFV的免疫胶体金检测方法,并对试纸条进行敏感性、特异性和重复性检测。【结果】SDS-PAGE结果显示,在IPTG终浓度为0.75 mmol/L时,37℃诱导8 h的条件下重组BEFV G蛋白表达量最大,且以包涵体形式表达,分子质量约为46 ku。Western blotting结果显示,浓缩纯化后的重组BEFV G蛋白具有较好的反应原性,可作为包被抗原用于免疫胶体金试纸条的建立。在质控线包被的抗体羊抗鼠IgG浓度为2 mg/mL,BEFV G蛋白包被浓度为0.6 mg/mL,喷金量为2.5μL/cm的条件下,3~5 min就可完成检测。制备的试纸条有较高的敏感性和特异性,血清稀释倍数为40倍的时候显色接近消线,且试纸条仅与BEFV阳性血清发生显色反应,与牛冠状病毒(BCoV)、牛轮状病毒(BRV)、牛传染性鼻气管炎病毒(IBRV)等其他病毒阳性血清均无交叉反应,阳性血清样本重复性试验证明试纸条的重复性较高,成功建立了免疫胶体金检测方法。【结论】本研究以原核表达的G蛋白为抗原成功制备了检测BEFV的新型免疫胶体金试纸条,该检测试纸条特异性强、稳定性好,适合用于临床快速检测BEF。【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 2.5μL/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 Bovin

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 

分 类 号：S852.63[农业科学—基础兽医学]