论文部分内容阅读
为制备抗发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)结构蛋白的单克隆抗体,本研究用灭活纯化的SFTSV病毒颗粒免疫BALB/c小鼠,利用杂交瘤技术获得分别分泌抗糖蛋白单抗和核蛋白单抗的杂交瘤细胞株。用免疫荧光法和免疫沉淀方法对制备的单克隆抗体的抗原特异性进行鉴定,并初步进行单抗效价、中和活性及亲和力等功能分析。结果显示,通过细胞融合和克隆化,共筛选出13株稳定分泌抗糖蛋白(Glycoprotein,GP)单抗和7株稳定分泌抗核蛋白(Nucleoprotein,NP)单抗的杂交瘤细胞株。免疫荧光和免疫沉淀鉴定显示获得的单抗有良好的抗原特异性。抗GP单抗中6株针对Gn,7株针对Gc,大部分的间接免疫荧光(Indirect immunofluorescence assay,IFA)滴度在1 280~20 480之间,其中4株抗Gn单抗具有中和活性。获得的7株抗NP单抗均与NP特异性结合,IFA滴度范围在5 120~20 480,均无中和活性。此外,经非竞争ELISA检测的两株抗GP单抗(1C8和1G8)均有较高亲和力。本研究为SFTS诊断方法的发展及SFTSV致病机制研究奠定了基础。
In order to prepare a monoclonal antibody against structural protein of Severe fever with thrombocytopenia syndrome (SFTSV), in this study, BALB / c mice were immunized with SFTSV virion purified inactivated and purified by hybridoma technique Hybridoma cell lines secreting anti-glycoprotein and nucleoprotein monoclonal antibodies. Immunofluorescence and immunoprecipitation methods were used to identify the antigen specificity of the prepared monoclonal antibodies, and initial functional analysis of monoclonal antibody titer, neutralizing activity and affinity. The results showed that 13 hybridoma cell lines secreting monoclonal anti-Glycoprotein (GP) monoclonal antibody and 7 monoclonal antibodies secreting stable anti-nuclear protein (NP) were screened by cell fusion and cloning. Immunofluorescence and immunoprecipitation identification showed that the McAbs obtained had good antigen specificity. Among the anti-GP monoclonal antibodies, 6 were against Gn and 7 were against Gc. Most of the IFA titers ranged from 1280 to 20 480, of which 4 anti-Gn mAbs had neutralizing activity . All of the 7 anti-NP monoclonal antibodies obtained were specifically bound to NP, and the IFA titer ranged from 5 120 to 20 480 with no neutralizing activity. In addition, both anti-GP monoclonal antibodies (1C8 and 1G8) tested by non-competitive ELISA showed higher affinity. This study lays the foundation for the development of SFTS diagnostic methods and the study of the pathogenesis of SFTSV.