Detection for CMV,LSV,LMoV Infected Lily with DNA Microarry Techniques

ZHU Chang-xiang; SONG Yun-zhi; WANG Mei-mei; WANG Xiu-fang; WEN Fu-jiang

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August 2005

ZHU Chang-xiang, SONG Yun-zhi, WANG Mei-mei, WANG Xiu-fang and WEN Fu-jiang. Detection for CMV,LSV,LMoV Infected Lily with DNA Microarry Techniques[J]. Virologica Sinica, 2005, 20(4): 434-437.

Citation: ZHU Chang-xiang, SONG Yun-zhi, WANG Mei-mei, WANG Xiu-fang, WEN Fu-jiang. Detection for CMV,LSV,LMoV Infected Lily with DNA Microarry Techniques .VIROLOGICA SINICA, 2005, 20(4) : 434-437.

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

朱常香 ^1,2 ,
宋云枝 ^1,2 ,
王玫玫 ¹ ,
王秀芳 ³ ,
温孚江 ^2*

1.
1. 山东泰安山东省作物生物学重点实验室 2.山东农业大学生命科学学院 3.中国农业科学院烟草研究所山东青州

摘要

烟草环斑病毒(Tobaccoringspotvirus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大。本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT-PCR扩增得到长约1500bp的目的片段。将目的片段与质粒pET-22b(+)连接,构建了含TRSVcp基因的融合蛋白原核表达载体pETRSV-CP。序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%~94.6%。将pETRSV-CP转入大肠杆菌,诱导表达。SDS-PAGE结果显示,表达的TRSVCP融合蛋白的相对分子质量约为58kDa。以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应。
- 烟草环斑病毒
- , 外壳蛋白基因
- , 原核表达
- , 抗血清

Detection for CMV,LSV,LMoV Infected Lily with DNA Microarry Techniques

Abstract

Coat protein (CP) gene of Tobacco ringspot virus (TRSV) Shandong isolate 1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR), and was subcloned into the pET-22b(+) prokaryotic expression vector. The recombinant vector was transformed into E.coli strain BL21. Sequence analysis revealed that the cp gene was 1548 nucleotides in length,encodes a coat protein of 515 amino acids, and shares 90.7%~94.6% nucleotides and amino acid homology with TRSV cp genes registered in GenBank. The target fusion peptide with a molecular weight of 58kDa was expressed under the condition of 23-25℃ and induced by IPTG at a final concentration of 1mmol/L. Rabbit was immunized using the expressed target peptide as antigen, and the antiserum was obtained. The antiserum had a titer of 1/1024 with high specificity to TRSV.
- Tobacco ringspot virus
- , Coat protein gene
- , Prokaryotic expression
- , Viral-specific antiserum

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Detection for CMV,LSV,LMoV Infected Lily with DNA Microarry Techniques

Abstract: Coat protein (CP) gene of Tobacco ringspot virus (TRSV) Shandong isolate 1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR), and was subcloned into the pET-22b(+) prokaryotic expression vector. The recombinant vector was transformed into E.coli strain BL21. Sequence analysis revealed that the cp gene was 1548 nucleotides in length,encodes a coat protein of 515 amino acids, and shares 90.7%~94.6% nucleotides and amino acid homology with TRSV cp genes registered in GenBank. The target fusion peptide with a molecular weight of 58kDa was expressed under the condition of 23-25℃ and induced by IPTG at a final concentration of 1mmol/L. Rabbit was immunized using the expressed target peptide as antigen, and the antiserum was obtained. The antiserum had a titer of 1/1024 with high specificity to TRSV.