华南农业大学学报2017，Vol.38Issue(1)：58-62,5.DOI:10.7671/j.issn.1001-411X.2017.01.010
南方水稻黑条矮缩病毒安徽分离物S5片段的克隆及S5-2基因的原核表达
Prokaryotic expression of S5-2 gene and cloning of S5 segment from Anhui isolate of Southern rice black-streaked dwarf virus
摘要
Abstract
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.关键词
南方水稻黑条矮缩病毒/S5片段/S5-2基因/原核表达Key words
Southern rice black-streaked dwarf virus/S5 segment/S5-2 gene/prokaryotic expression分类
农业科技引用本文复制引用
江彤,单文书,夏伟伟,张享享,蒋西子..南方水稻黑条矮缩病毒安徽分离物S5片段的克隆及S5-2基因的原核表达[J].华南农业大学学报,2017,38(1):58-62,5.基金项目
国家自然科学基金(31371915) （31371915）
公益性行业(农业)科研专项(201303028) （农业）
