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国家高技术研究发展计划(2007AA021505)

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水稻矮缩病毒非结构蛋白Pns6的表达及其单克隆抗体的特异性分析被引量:1
2009年
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.
吉栩魏春红李毅
关键词:水稻矮缩病毒单克隆抗体抗原决定簇
Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies
2009年
The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th-509th amino acids) of Pns6, which is confirms bioinformatics analysis.
JI Xu, WEI ChunHong & LI Yi Peking–Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, the National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China
关键词:RICEDWARFEXPRESSIONMONOCLONALANTIGENDETERMINANT
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