摘要
利用大肠杆菌表达系统表达裂谷热病毒(Rift Valley fever virus,RVFV)Gn蛋白Ⅱ-Ⅲ结构域的重组蛋白作为包被抗原,建立检测RVFV抗体的间接ELISA方法。将RVFV Gn蛋白Ⅱ-Ⅲ结构域基因序列克隆至pET-30a(+),构建重组质粒pET-RVFV Gn-DⅡ-Ⅲ,并将其转化至DE3(BL21)感受态细菌,用IPTG诱导表达重组Gn-DⅡ-Ⅲ蛋白。以纯化的重组蛋白作为包被抗原,SPA-HRP为酶标二抗建立检测RVFV抗体的间接ELISA方法。Western blot鉴定结果证明RVFV Gn蛋白DⅡ-Ⅲ成功表达;进一步确定最佳诱导表达条件为0.8 mmol/L IPTG,37℃诱导5 h。利用亲和层析法纯化Gn-DⅡ-Ⅲ蛋白,纯度达91.9%,以纯化后蛋白作为包被抗原建立RVFV的间接ELISA抗体检测方法。特异性试验结果显示,该方法只能检测出RVFV阳性血清,与西尼罗病毒(West Nile virus,WNV)、埃博拉病毒(Ebola virus,EBOV)、马尔堡病毒(Marburg virus,MARV)和蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)阳性血清均无交叉反应。敏感性试验结果显示,RVFV Gn-DⅢ-Ⅲ阳性血清稀释至6400倍时检测结果仍为阳性,表明该方法具有良好的敏感性。重复性试验结果显示,批内和批间反应的变异系数均小于10%,表明该方法具有良好的重复性。本研究利用表达与纯化的Gn-DⅡ-Ⅲ蛋白作为包被抗原,建立了检测RVFV抗体的间接ELISA方法,为RVFV检测方法与新型疫苗的研发奠定了基础。
This study aims to establish an indirect ELISA method for detecting RVFV antibodies u-sing recombinant proteins of Rift Valley fever virus(RVFV)Gn proteinⅡ-Ⅲstructural domains as the encapsulated antigen which was expressed by the Escherichia coli(E.coli)expression sys-tem.The gene sequences encoding theⅡandⅢsubdomains of RVFV Gn protein were inserted in-to pET-30a(+)to construct the recombinant plasmid pET-RVFV Gn-DⅡ-Ⅲ.After transforma-tion of the recombinant plasmid into DE3(BL21)competent cells,the recombinant Gn-DⅡ-Ⅲprotein was induced with IPTG and purified using affinity chromatography.An indirect ELISA method for the detection of RVFV antibodies was developed using purified recombinant protein as coating antigen and SPA-HRP as the enzyme-labelled secondary antibody.Western blot analysis confirmed that the RVFV Gn-DⅡ-Ⅲprotein was successfully expressed.The optimal expression conditions for RVFV Gn-DⅡ-Ⅲprotein were induced with 0.8 mmol/L IPTG at 37℃for 5 h.The Gn-DⅡ-Ⅲprotein was purified using affinity chromatography with a purity of 91.9%,and the purified protein was used as the encapsulated antigen to develop an ELISA assay for RVFV anti-bodies.The specificity evaluation showed that the method specifically detected RVFV-positive sera and did not cross-react with sera positive for West Nile virus(WNV),Ebola virus(EBOV),Mar-burg virus(MARV)and tick-borne encephalitis virus(TBEV).When the RVFV Gn-DⅢ-Ⅲposi-tive serum was diluted to 6400 times,the test result still showed positive results,demonstrating the method had good sensitivity.The repeatability evaluation results indicated that the variation co-efficients for both intra-and inter-batch responses was less than 10%,indicating that the method had good repeatability.In conclusion,the RVFV Gn-DⅡ-Ⅲprotein was successfully expressed u-sing the E.coli expression system.The purified recombinant Gn-DⅡ-Ⅲprotein was used as the encapsulated antigen to develop an indirect ELISA assay for RVFV antibodies,which provides a preliminary basis for the diagnosis of RVF and the research and development of RVF vaccines.
作者
栾娇彦
张梦瑶
焦翠翠
张向阳
艾丽思
黄培
李媛媛
张海丽
王化磊
LUAN Jiaoyan;ZHANG Mengyao;JIAO Cuicui;ZHANG Xiangyang;AI Lisi;HUANG Pei;LI Yuanyuan;ZHANG Haili;WANG Hualei(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases,College of Veterinary Medicine,Jilin University,Changchun 130062,China)
出处
《中国兽医学报》
北大核心
2025年第6期1186-1193,1209,共9页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2021YFF0703600)。
作者简介
栾娇彦(2000-),女,硕士研究生;通信作者:王化磊,E-mail:wanghualei@jlu.edu.cn。
