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  

Prokaryotic expression of protein encoded by gene S7-1 and cloning of genome S7 of Anhui isolate of Southern rice black-streaked dwarf virus

作　　者：江彤[1] 何志龙[1] 张享享 夏伟伟[1] 李祥宇[1] 

机构地区：[1]安徽农业大学植物保护学院,合肥230036

出　　处：《植物保护学报》2017年第3期467-472,共6页Journal of Plant Protection

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)

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。To clarify the population variation of Southern rice black-streaked dwarf virus（SRBSDV）and obtain prokaryotic expression protein of S7-1, the genome S7 of SRBSDV Anhui isolate was amplified by RT-PCR, and then the genome were cloned, sequenced and analyzed. S7-1 gene of SRBSDV Anhui isolate was further amplified using specific primers and cloned into the prokaryotic expression of the vector pET-GST. Recombinant plasmid pET-S7-1 was transformed into Escherichia coli BL21（DE3）. Fusion protein was induced with IPTG and purified with Ni2＋-NTA affinity column, and was detected with Western blot assay. The results showed that S7 of SRBSDV Anhui isolate shared higher sequence similarity with 99.3%-99.9% to that of other isolates of SRBSDV, while S7 of SRBSDV Anhui isolate had relatively lower sequence similarity with 35.5%-73.3% to that of other species of Fijivirus.The SRBSDV Anhui isolate and other isolates of SRBSDV were clustered into a separate branch, and the relationship among the isolates of SRBSDV was much closer. The fusion protein S7-1 with an approximate molecular weight of 68 kD was obtained, and the GST monoclonal antibody could specifically bind to the fusion protein S7-1, which indicated that the fusion protein was indeed the target protein.

关 键 词：南方水稻黑条矮缩病毒 S7片段 克隆 S7-1基因 原核表达 

分 类 号：S435.11[农业科学—农业昆虫与害虫防治]