【目的】探讨南方水稻黑条矮缩病毒( Southern rice black-streaked dwarf virus,SRBSDV)安徽分离物( SRBSDV-AnHui-HN2)的遗传特性,并获得原核表达的P5-2蛋白。【方法】RT-PCR扩增SRBSDV S5片段,克隆、测序并进行序列分析。将S5-2基因插入原核表达载体,重组载体转化大肠埃希菌并用IPTG诱导,Ni2+-NTA亲和柱纯化融合蛋白,SDS-PAGE分析P5-2蛋白的表达情况。【结果】SRBSDV-AnHui-HN2 S5片段全长3167 bp,包含S5-2基因全长612 bp,编码204个氨基酸。序列比对结果显示,SRBSDV-AnHui-HN2 S5片段与其他SRBSDV分离物S5片段的序列相似性极高,达99．0%~99．7%,而与斐济病毒属( Fijivirus)其他成员S5片段的序列相似性较低,仅为38．0%~71．3%；构建的S5片段系统发育树表明SRBSDV和RBSDV聚成1个分支,其中6个SRBSDV分离物聚成1个亚分支。原核表达获得相对分子质量约为47000的重组蛋白,Western blot分析显示,GST单抗能够与重组融合蛋白发生特异性反应。【结论】SRBSDV各分离物之间亲缘关系非常近,而与Fijivirus其他成员亲缘关系较远,原核表达获得的融合蛋白为靶标蛋白。%Objective] To study genetic characteristics of the isolates of Southern rice black-streaked dwarf virus ( SRBSDV ) from Anhui province, and to obtain P5-2 protein by prokaryotic expression.[Method]The S5 segment of SRBSDV was amplified by RT-PCR, and it was cloned, sequenced and analyzed. Gene S5-2 was inserted into prokaryotic expression vector. The recombinant vector was transformed into Escherichia coli and was induced by IPTG. The fusion protein was purified by Ni2+-NTA affinity column. The expression of P5-2 protein was analyzed by SDS-PAGE. [Result] The S5 segment from Anhui isolate of SRBSDV(SRBSDV-AnHui-HN2) was 3 167 bp in full length and contained a 612 bp S5-2 gene encoding 204 amino acids. Sequence comparison showed that the S5 segment of SRBSDV-AnHui-HN2 shared high sequence similarity(99. 0% -99. 7%) with other SRBSDV isolates, while had relatively low sequence similarity ( 38. 0% -71. 3%) to other Fijivirus members. The phylogenetic tree based on S5 segment sequences showed that SRBSDV and RBSDV clustered into a branch, and six isolates of SRBSDV clustered into a sub-branch. The recombinant protein with approximately 47 000 relative molecular mass was obtained by prokaryotic expression. Western blot analysis revealed that GST monoclonal antibody could specifically bind to the fusion protein. [Conclusion]All isolates of SRBSDV are closely related, and they have relatively far relationship to other Fijivirus members. The fusion protein obtained by prokaryotic expression is the target protein.
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