Preparation and application of antiserum against watermelon mosaic virus coat protein expressed in E. coli.
Preparation and application of antiserum against watermelon mosaic virus coat protein expressed in E. coli.
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DOI：
10.13926/j.cnki.apps.000237
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发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Tian, Y. P.
中科院分区：
文献类型：
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作者：
Ji, Shu-Xian;Wang, Shu-Wen;Tian, Y. P.
西瓜花叶病毒（Watermelon mosaic virus，WMV）是一种危害瓜类作物的重要病毒。制备高特异性、高灵敏度的抗血清是快速、准确检测和鉴定WMV的关键。本研究将WMV外壳蛋白（CP）基因克隆到E.大肠杆菌表达载体pEHISTEV-WMV-CP。将pEHISTEV-WMV-CP转化E. coli Rosetta。经IPTG诱导后，获得了一个预期分子量为36 kDa的蛋白。剪下免疫新西兰白色兔。Westernblotting结果表明，该抗血清能特异性识别WMV CP蛋白。coli和WMV。与马铃薯Y病毒属的番木瓜环斑病毒（PRSV）、马铃薯Y病毒（PVY）、小西葫芦黄化花叶病毒（ZYMV）和烟草花叶病毒属的黄瓜绿色斑驳花叶病毒（CGMMV）均无交叉反应。该抗血清经PTA-ELISA检测效价为1：8192，可检测稀释512倍的WMV感染样品。在RT-PCR阳性的10份样品中，用该抗血清进行PTA-ELISA检测，均为阳性。结果表明，所制备的抗血清具有较高的特异性和敏感性，为WMV的快速、准确检测和鉴定奠定了基础。
Watermelon mosaic virus (WMV; genus Potyvirus) is an economically important virus infecting cucurbit crops. Preparation of antiserum with high specificity and sensitivity is essential for rapid and accurate detection and identification of WMV. In this study, WMV coat protein (CP) gene was cloned into E. coli expression vector pEHISTEV, named pEHISTEV-WMV-CP. pEHISTEV-WMV-CP was transformed and expressed into E. coli Rosetta. A protein with expected molecular weight of 36 kDa was obtained after IPTG induction. The band was cut and used to immune New Zealand white rabbit. Western blotting results showed that the antiserum could specifically recognize WMV CP expressed in E. coli and WMV from infected leaves. There was no cross reaction to closely related Papaya ringspot virus (PRSV), Potato virus Y (PVY), Zucchini yellow mosaic virus (ZYMV) from the genus Potyvirus, neither the Cucumber green mottle mosaic virus (CGMMV) of the genus Tobamovirus. The antiserum had a titer of 1:8 192 in PTA-ELISA and could detect WMV infected samples diluted up to 512 times. Among the 10 samples showed positive reaction in RT-PCR, all of them were positive when detected with this antiserum via PTA-ELISA. These results indicated that the antiserum obtained in our study had high specificity and sensitivity and laid solid foundation for rapid and accurate detection and identification of WMV.