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水稻矮缩病毒(RDV)的基因组包含12条双链RNA,其中基因组片段S6编码的非结构蛋白Pns6为该病毒的运动蛋白.在本研究中,在大肠杆菌中表达了N端融合His-tag的重组Pns6蛋白.在低温和低IPTG浓度的诱导后,通过Ni螯合亲和层析进行纯化获得了HisPns6蛋白.稳定性分析表明,HisPns6是一个稳定的蛋白,可耐受37℃下长达24h的处理.纯化的蛋白后续用于单克隆抗体的制备并得到18个杂交瘤细胞株系.使用从感染RDV的水稻叶片中提取的Pns6为样品,以健康水稻总蛋白为对照,通过Western blot法对抗体进行特异性分析,获得了15个阳性抗体.对其进行抗原决定簇分析表明,最敏感的抗原决定簇位于Pns6的C端区域(296~509位氨基酸).该结果与生物信息学预测分析相吻合.
The genome of rice dwarf virus (RDV) contains 12 double-stranded RNAs, of which the non-structural protein Pns6 encoded by genome segment S6 is the motor protein of the virus.In this study, N-terminal fusion His-tag was expressed in E. coli Of recombinant Pns6 protein HisPns6 protein was purified by Ni chelating affinity chromatography after induction at low temperature and low IPTG concentration Stability analysis showed that HisPns6 is a stable protein that can tolerate up to 37 ℃ 24h.The purified protein was used for the preparation of monoclonal antibody and 18 hybridoma cell lines were obtained.Pns6 extracted from rice leaves infected with RDV was used as a sample and healthy rice total protein was used as a control and analyzed by Western blot The results showed that the most sensitive antigenic determinant was located in the C-terminal region of Pns6 (amino acids 296-509) .The results were consistent with bioinformatics Forecast analysis is consistent.