蜱媒Dabieshan病毒NP蛋白的原核表达及间接ELISA检测方法的建立  

AN INDIRECT ELISA ANTIBODY SCREEN METHOD AGAINST DABIESHAN VIRUS VIA RECOMBINANT NP COATING ANTIGEN

作　　者：何婷 艾乐乐 朱长强 胡丹 王长军 李萌[1] 谭伟龙 HE Ting;AI Le-Le;ZHU Chang-Qiang;HU Dan;WANG Chang-Jun;LI Meng;TAN Wei-Long(Department of Blood Transfusion,Children’s Hospital of Nanjing Medical University,Nanjing 210000,China;Eastern Theater CDC,Nanjing 210000,China)

机构地区：[1]南京医科大学附属儿童医院输血科,江苏南京210000 [2]东部战区疾病预防控制中心,江苏南京210000

出　　处：《寄生虫与医学昆虫学报》2021年第3期184-188,共5页Acta Parasitologica et Medica Entomologica Sinica

基　　金：江苏省333工程科研项目(BRA2018410);国家自然科学基金项目(U160223);江苏省卫建委项目基金(M2020087)。

摘　　要：为建立蜱媒Dabieshan病毒血清学抗体检测方法,本研究基于Dabieshan病毒NP蛋白序列,通过建立原核表达体系并优化反应条件,建立一种可用于检测血清NP抗体的间接ELISA方法。结果显示,当重组NP蛋白包被浓度为0.23μg/孔,血清稀释比例为1∶5×10^(4),二抗稀释比例为1∶5000,底物显色时间为15 min时,抗原抗体反应强度最佳。以上述条件建立的间接ELISA方法,与同为白蛉病毒属的发热伴血小板减少综合征病毒(SFTSV)阳性血清无交叉反应,具有较好的特异性。批内OD_(450)值变异系数小于10%,重复性较好。结果表明,本研究建立的间接ELISA方法具有较高的特异性和重复性,可进一步用于Dabieshan病毒携带区的人群及动物血清筛查,为了解该病毒的潜在致病性提供流行病学资料。To establish an indirect ELISA method to screen the prevalence of Dabieshan virus in populations,a recombinant NP coating antigen of Dabieshan virus was expressed and purified in prokaryote expression system.With the purified recombinant NP antigen,the indirect ELISA method was developed and optimized via specificity and repeatability tests.Results showed that the intensity of antigen antibody reaction was the best when the dilutions of serum and HRP-labeled secondary antibody were 1∶5×10^(4) and 1∶5000 respectively,0.23μg NP antigen coated in each well,and the reaction time with TMB was optimized as 15 min.The high specificity and good repeatability were also shown in the indirect ELISA method established under the above conditions with no cross-reaction with SFTSV-positive serum and less than 10%variation with batch.An indirect ELISA method with high specificity and repeatability has been established in the present study,which may be used in screening Dabieshan virus in populations and animals,and will help us to understand the potential pathogenesis.

关 键 词：Dabieshan病毒 原核表达 NP蛋白 间接ELISA 

分 类 号：R373[医药卫生—病原生物学]