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

发布时间：2018-02-24 16:50

  本文关键词： 南方水稻黑条矮缩病毒 S片段 S-基因 原核表达　出处：《华南农业大学学报》2017年01期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] to study the genetic characteristics of Southern rice black-streaked dwarf virus (SRBSDV) isolated from Anhui Province, and obtain the P5-2 protein expressed in prokaryotic expression. [methods] SRBSDV S5 fragment was amplified by RT-PCR and cloned. S5-2 gene was inserted into prokaryotic expression vector. The recombinant vector was transformed into Escherichia coli and purified by IPTG induced Ni2-NTA affinity column. The expression of P5-2 protein was analyzed by SDS-PAGE. [results] the total length of SRBSDV-An Hui-HN2 S5 fragment was 3 167 BP, and the S5-2 gene was 612 BP, encoding 204 amino acids. The results showed that the sequence similarity between SRBSDV-An Hui-HN2 S5 fragment and other SRBSDV isolate S5 fragment was very high. 99.0 and 99.7, and the sequence of S5 fragments of Fijivirus, other members of Fijivirus, is less similar to that of S5 fragments, which are only 38.0% and 71.3%. The phylogenetic tree of S5 fragments constructed shows that SRBSDV and RBSDV are clustered into one branch. Among them, 6 SRBSDV isolates were clustered into one subbranch. Western blot analysis of the recombinant protein with relative molecular weight of 47 000 showed that the McAb could react specifically with the recombinant fusion protein. [conclusion] the SRBSDV isolates have specific reaction with the recombinant fusion protein. [conclusion]. Are very close to each other, The fusion protein expressed in prokaryotic expression was the target protein.
【作者单位】： 安徽农业大学植物保护学院;
【基金】：国家自然科学基金(31371915) 公益性行业(农业)科研专项(201303028)
【分类号】：S435.111.4
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本文编号：1530996