Prokaryotic expression of S5-2 gene and cloning of S5 segment from Anhui isolate of Southern rice black-streaked dwarf virus
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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（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-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。
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关键词:
- 南方水稻黑条矮缩病毒 /
- S5片段 /
- S5-2基因 /
- 原核表达
Abstract:ObjectiveTo 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.
MethodThe 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.
ResultThe 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.
ConclusionAll 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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图 1 基于S5片段核苷酸序列构建的系统关系树
Figure 1. Relationship dendrogram of SRBSDV-Anhui-HN2 and other isolates of Fijivirus based on S5 segment sequences
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图 2 重组质粒pET-S5-2的酶切电泳图谱
M：DNA marker DL2000；1：BamH I和Sal I双酶切pET-S5-2；2：BamH I单酶切pET-S5-2；3：Sal I单酶切pET-S5-2；4：重组质粒pET-S5-2。
Figure 2. Electrophoresis pattern of recombinant plasmid pET-S5-2 digested with restriction enzyme
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图 3 融合蛋白的SDS-PAGE和Western blot分析
M：分子质量蛋白Marker；1：pET-GST空载体未诱导；2：pET-GST空载体诱导；3：pET-S5-2未诱导；4：pET-S5-2诱导；5：纯化的融合蛋白。
Figure 3. SDS-PAGE and Western blot analysis of fusion protein
表 1 SRBSDV-Anhui-HN2 S5片段与16个斐济病毒属其他分离物S5片段核苷酸序列的相似性
Table 1 Nucleotide sequence similarity between S5 segment of SRBSDV-Anhui-HN2 and S5 segments of 16 other isolates of Fijivirus
分离物 GenBank登录号 来源地 相似性/% SRBSDV-Anhui-HN2 HF954999 中国安徽 100.0 SRBSDV-VNM JQ692576 越南 99.7 SRBSDV-HuNyy JQ034352 中国湖南 99.5 SRBSDV-Hubei HM585275 中国湖北 99.4 SRBSDV-YN-MShi JQ773424 中国云南 99.2 SRBSDV-HN FN563993 中国海南 99.0 RBSDV-HB KC134293 中国河北 71.3 RBSDV-JS KM921677 中国江苏 71.1 RBSDV-SDZZ10 JX421768 中国山东 70.2 RBSDV-MX8 HF954989 中国安徽 70.2 RBSDV-Anhui-LJ HF955009 中国安徽 70.2 RBSDV-WZ KC801047 中国浙江 70.1 RBSDV-LH KC801046 中国浙江 70.0 RBSDV-Korean HQ670667 韩国 69.9 FDV-AUS NC_007160 澳大利亚 49.9 MRCV-Arg AY607587 阿根廷 64.1 NLRV-TSU D49697 古巴 38.0 
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