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

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pET-GST上，重组质粒pET-S7-1转化大肠杆菌BL21（DE3），IPTG诱导及Ni2+-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 kD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 SRBSDVAnhui isolate was amplified by RT-PCR,and then the genome were cloned,sequenced and analyzed.S7-1 gene of SRBSDVAnhui 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.