南方水稻黑条矮缩病毒安徽分离物S7片段的克隆及其S7-1基因编码蛋白的原核表达

发布时间：2018-12-06 07:29

【摘要】：为探讨南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的种群变异并获得S7-1原核表达蛋白,采用RT-PCR技术扩增SRBSDV安徽分离物S7片段,并进行克隆、测序及序列分析,再利用特异性引物扩增该分离物S7-1基因并克隆到原核表达载体p ET-GST上,重组质粒p ET-S7-1转化大肠杆菌BL21(DE3),IPTG诱导及Ni~(2+)-NTA亲和柱纯化融合蛋白,再进行Western blot检测。结果显示,SRBSDV安徽分离物S7片段与其它SRBSDV各个分离物S7片段的序列相似性极高,达99.3%~99.9%,而与斐济病毒属Fijivirus其它种之间的序列相似性较低,为35.5%~73.3%。SRBSDV安徽分离物与其它SRBSDV各个分离物聚成1个单独的分支,彼此间亲缘关系较近。试验获得了分子质量约为68 k D的S7-1融合蛋白,且GST单抗能够与S7-1融合蛋白发生特异性反应,表明本研究得到的融合蛋白确为靶标蛋白。
[Abstract]:In order to study the population variation of southern rice black stripe dwarf virus (Southern rice black-streaked dwarf virus,SRBSDV) and obtain the prokaryotic expression protein S7-1, the S7 fragment of SRBSDV Anhui isolate was amplified by RT-PCR technique, and cloned, sequenced and sequenced. The S7-1 gene was amplified by specific primers and cloned into prokaryotic expression vector p ET-GST. The recombinant plasmid p ET-S7-1 was transformed into Escherichia coli BL21 (DE3), IPTG induced) and purified by Ni~ (2) -NTA affinity column. Western blot test was performed. The results showed that the sequence similarity between S7 fragment of Anhui isolate of SRBSDV and that of other SRBSDV isolates was very high, up to 99.39.The sequence similarity between S7 fragment of Anhui isolate and other species of Fijian virus Fijivirus was lower than that of other species of Fijian virus. 35.5%~73.3%.SRBSDV Anhui isolate and other SRBSDV isolates were clustered into a single branch, and the relationship between them was close. The S7-1 fusion protein with molecular weight of about 68kD was obtained, and the GST monoclonal antibody could react specifically with S7-1 fusion protein, which indicates that the fusion protein obtained in this study is a target protein.
【作者单位】：安徽农业大学植物保护学院;
【基金】：国家自然科学基金(31371915,31671999) 安徽省教育厅自然科学基金(KJ2012A113)
【分类号】：S435.11

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