发热伴血小板减少综合征布尼亚病毒核衣壳蛋白单克隆抗体的制备及初步应用

The preparation and primary application of mAbs against nucleocapsid protein of severe fever with thrombocytopenia syndrome virus

目的制备发热伴血小板减少综合征布尼亚病毒(SFTSV)核衣壳蛋白(NP)的单克隆抗体,并探讨其在该病诊断中的作用。方法克隆NP基因,构建原核表达载体pComb3XSS-NP,并转化大肠杆菌Top10F′,经诱导表达,纯化获得6HIS-NP融合蛋白。以该融合蛋白免疫Balb/c小鼠,经杂交瘤技术制备NP的鼠源单克隆抗体。经鉴定后,选择1株1C8-C6进行大量制备、纯化,并偶联辣根过氧化物酶(HRP),用该酶标抗体对SFTSV感染人的急性期血清进行Western blot检测。结果成功表达、纯化SFTSV重组的NP。经小鼠免疫、细胞融合和ELISA筛选后,共获得4株鼠源单克隆抗体。具较高滴度的1C8-C6酶标抗体能与SFTSV感染人的急性期血清中的NP在Western blot水平上结合,而与汉坦病毒、登革热病毒感染人急性期血清及正常人血清不反应。结论 SFTSV NP的表达及其单抗的制备为该病的实验室诊断奠定了基础。

The recent emergence of the confirmed human infection of severe fever with thrombocytopenia syndrome virus（SFTSV） in China is of global concern and causes high fatality.The clinical diagnosis is urgently needed to differentiate the disease from other infections.In this study,we aim to develop mAbs against SFTSV nucleocapsid protein（NP）,and explored their application in disease diagnosis.Gene encoding NP was amplified from SFTSV genome by RT-PCR,and then cloned into prokaryotic expression vector pComb3XSS.The recombinant expression vector was expressed in Top10F′ E.coli.host,and the recombinant protein was purified with its fusion partner 6HIS tag by affinity chromatography.Then Balb/c mice were immunized with NP for monoclonal antibodies（mAbs） production.Of the four positive mAbs clones screened by ELISA,one positive mAb clone designated 1C8-C6 with the highest titre was selected for manufacturing,purification and horseradish peroxidase（HRP） coupling.Western blot has been designed for detection of NP from SFTSV-infected human acute phase sera using 1C8-C6-HRP conjugate.The result showed that the mAb could effectively bind NP in human SFTSV-infected viremia sera,with no cross-reactivity to hanta,dengue virus-infected acute phase sera,and healthy human sera.The NP expression and its mAbs development provide the robust basis for disease diagnosis.