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

【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（SRBSDVAn Hui-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-An Hui-HN2 S5片段全长3 167 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,SRBSD...

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Published in华南农业大学学报 Vol. 38; no. 1; pp. 58 - 62
Main Author 江彤 单文书 夏伟伟 张享享 蒋西子
Format Journal Article
LanguageChinese
Published 安徽农业大学 植物保护学院,安徽 合肥,230036 2017
Subjects
S5-2基因
S5片段
南方水稻黑条矮缩病毒
原核表达
S5-2基因
S5 segment
S5-2 gene
S5片段
原核表达
Southern rice black-streaked dwarf virus
prokaryotic expression
南方水稻黑条矮缩病毒
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ISSN1001-411X
DOI10.7671/j.issn.1001-411X.2017.01.010

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Summary:【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（SRBSDVAn Hui-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-An Hui-HN2 S5片段全长3 167 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-An Hui-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47 000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。
Bibliography:Southern rice black-streaked dwarf virus; S5 segment; S5-2 gene; prokaryotic expression
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-An Hui-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 SRBSDVAn Hui-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 phylogene
ISSN:1001-411X
DOI:10.7671/j.issn.1001-411X.2017.01.010