Expression of nucleocapsid protein of capsicum chlorosis virus in a prokaryotic system and its application in virus detection.
Expression of nucleocapsid protein of capsicum chlorosis virus in a prokaryotic system and its application in virus detection.
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DOI：
10.4454/jpp.v96i2.032
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发表时间：
2014-07
影响因子：
2.2
通讯作者：
X. Rao;Yuanyou Li;Zilin Wu;Yingliang Liu;Wence Wang
中科院分区：
文献类型：
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作者：
X. Rao;Yuanyou Li;Zilin Wu;Yingliang Liu;Wence Wang
辣椒褪绿病毒（Capsicum chlorosis virus，CaCV）属于番茄斑萎病毒属西瓜银斑驳病毒（Watermelon Silver Mottle Virus，WSMoV）血清群，可严重危害重要经济作物。利用RT-PCR技术对一株来自蝴蝶兰的CaCV（CaCV-pha）进行了鉴定。CaCV-pha核衣壳蛋白（NP）的氨基酸序列与其它CaCV分离物的同源性为92.0%~ 97.1%，与其它CaCV分离物的同源性为92.0%~ 97.1%。由于CaCV感染的作物的鉴定需要敏感的诊断工具的可用性，使用原核表达系统来获得用于抗血清生产的重组CaCV-pha NP。将CaCV-phaNP基因亚克隆到pET-28 a（+）载体中，转化大肠杆菌Rosetta（DE 3）细胞进行表达。纯化后，将重组蛋白注射到兔体内以产生多克隆抗血清，在Western印迹试验中，所述多克隆抗血清与用作抗原的重组蛋白强烈反应。抗CaCV-NP血清的可用性将有助于对CaCV的实地调查和进一步研究。
Capsicum chlorosis virus (CaCV), which belongs to the Watermelon silver mottle virus (WSMoV) serogroup of the genus Tospovirus, can seriously harm economically important crops. A CaCV strain from Phalaenopsis orchids (CaCV-pha) was identified by RT-PCR. The amino acid sequence of the CaCV-pha nucleocapsid protein (NP) shared 92.0% to 97.1% identity with comparable proteins of other CaCV isolates, with which it was phylogentically related. Since the identification of CaCV-infected crops requires the availability of sensitive diagnostic tools, a prokaryotic expression system was used to obtain a recombinant CaCV-pha NP for antiserum production. The CaCV-pha NP gene was subcloned into the pET-28a (+) vector and transformed into Escherichia coli Rosetta (DE3) cells for expression. After purification, the recombinant protein was injected into rabbits to generate a polyclonal antiserum which, in Western blot tests reacted strongly with the recombinant protein used as antigen. The availability of an anti-CaCV-NP serum will facilitates field surveys for, and further research on CaCV.