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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (22): 276-281.doi: 10.11924/j.issn.1000-6850.2014-0320

所属专题： 生物技术； 园艺

• 植物保护 农药 • 上一篇    下一篇

瓜类黄化褪绿病毒上海分离物外壳蛋白的原核表达及其抗血清的制备

曾蓉 徐丽慧 陆金萍 戴富明   

  • 收稿日期:2014-02-12 修回日期:2014-03-19 出版日期:2014-08-05 发布日期:2014-08-05
  • 基金资助:
    上海市农业科学院院发基金“葫芦科植物褪绿黄化病毒的抗血清制备及血清学检测方法的建立”[农科发2011(12)]；上海市科委科研计划项目“长三角地区主要农作物重大病虫害预测预报和生态工程治理”(13395810101)；上海市现代农业产业技术体系建设专项“上海市西瓜甜瓜产业技术体系建设”(2010-2015)；上海市科技兴农重点攻关项目“农业生物灾害监测预警及防控技术研究”[沪农科攻字(2012)第2-10号]；上海市科技兴农推广项目“优质哈密瓜标准化生产技术示范推广”[沪农科推字(2009)第5-6号]。

Prokaryotic Expression of Coat Protein of Cucurbit Chlorotic Yellows Virus and Its Antiserum Preparation

  • Received:2014-02-12 Revised:2014-03-19 Online:2014-08-05 Published:2014-08-05

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摘要： 利用RT-PCR技术扩增得到瓜类黄化褪绿病毒(cucurbit chlorotic yellows virus，CCYV)上海市嘉定分离物的外壳蛋白(coat protein，cp)。序列分析结果表明，cp基因的核苷酸序列长度为753 bp，编码250个氨基酸，与已报道的CCYV其他分离物的cp核苷酸相似性均高于95%。将此基因克隆到原核表达载体pET32a(+)上，转化大肠杆菌BL21(DE3)后IPTG进行诱导表达，SDS-PAGE及Western blotting分析表明，CCYV cp在大肠杆菌中可表达出含有6-His标签的分子量约46 kDa的蛋白。纯化表达蛋白后免疫家兔制备抗血清，间接酶联免疫吸附测定(ID-ELISA)结果显示，制备的抗血清可用于检测田间感病样品中的CCYV。

关键词: 增殖培养, 增殖培养

Abstract: The coat protein gene of a Chinese isolate of cucurbit chlorotic yellows virus was amplified by RTPCR. Sequence analysis showed that the cp gene was 753 bp, encoding 250 amino acid residues and exhibited high sequence similarities (over 95%) with all the CCYV reported in GenBank. The CCYV cp gene was cloned into the prokaryotic expression vector pET32a(+ ), then transformed into Escherichia coli BL21(DE3), and subsequently induced by IPTG. It was shown that the recombinant cp gene was expressed with the molecular weight of 46 kDa with 6-His tag by SDS-PAGE and Western blotting analysis. Antiserum was prepared after the rabbits were immunized with purified recombinant CCYV coat protein. Results showed that the antiserum specifically detected against CCYV in infected melon leaves by indirect enzyme-linked immunosorbent assay (ID-ELISA).