与烟草曲茎病毒相伴随的DNAβ的βC1基因原核表达及蛋白的单抗制备

Prokaryotic expression of βC1 gene of DNAβ satellite associated with Tobacco curly shoot virus and preparation of monoclonal antibodies against its protein

将PCR扩增的烟草曲茎病毒（Tobacco curly shoot virus，TbCSV）Y35分离物βC1基因克隆到pET-32a原核表达载体，构建原核表达重组载体pET32a-Y35βC1．重组载体导入大肠杆菌BL21（DE3），经过IPTG诱导、Ni^2＋ NTA亲和柱纯化获得与硫氧还蛋白融合的重组蛋白Y35βC1．以Y35βC1重组蛋白为抗原免疫BALB／c小鼠，用杂交瘤细胞技术制备了9株能够稳定传代并分泌抗Y35βC1蛋白单克隆抗体的杂交瘤细胞株，分别制备它们的单抗腹水．9株单抗腹水的间接ELISA效价在10^-5～10^-6之间.Y35βC1单克隆抗体的研制为该蛋白的亚细胞定位及功能研究，明确Y35βC1基因在致病过程中的作用机理奠定了基础．

The βC1 gone of DNAβ satellite associated with Tobacco curly shoot virus （TbCSV） isolate Y35 was obtained by PCR and was cloned into pET-32a to construct recombinant prokaryotic expression vector, pET32a-Y35βC1. The recombinant vector was then transformed into Escherichia coli BL21 （DE3）. Fusion protein trxA-Y35βC1 was induced by IPTG and purified with Ni^2＋ NTA affinity chromatography. Nine hybridoma cell lines secreting monoclonal antibodies （MAbs） against Y35βC1 were produced by fusing mouse myeloma cells （SP2/0） with spleen cells from BALB/c mice immunized with the recombinant protein. The titres of ascitic fluids of the nine MAbs ranged from 10^-5 to 10^-6 in indirect EIASA. The preparation of MAbs would be useful for sub cellular location and functional study of βC1 protein.