| 葡萄卷叶伴随病毒3号抗血清的制备及应用 |
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| 引用本文： | 王克双,费菲,吕睦由页,李捷,程玉琴.葡萄卷叶伴随病毒3号抗血清的制备及应用[J].中国农业大学学报,2012,17(4):81-85. |
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| 作者姓名： | 王克双 费菲 吕睦由页 李捷 程玉琴 |
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| 作者单位： | 中国农业大学农学与生物技术学院果树系／北京市果树逆境生理实验室,北京,100193 |
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| 基金项目： | 现代农业产业技术体系建设专项 |
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| 摘 要： | 为获得特异性抗血清用于批量检测葡萄苗木中的葡萄卷叶伴随病毒3号(Grapevine leafroll associatedvirus 3,GLRaV-3),本研究用含有GLRaV-3CP蛋白基因的载体pMD18-CP为模板,PCR扩增CP蛋白基因。将PCR产物定向克隆到原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3),筛选得到阳性克隆pET-G3CP。用终浓度为1mmol/L的IPTG进行诱导表达,SDS-PAGE电泳分析显示,GLRaV-3CP在大肠杆菌中得到高效表达。纯化表达产物后免疫家兔制备抗血清。经分析,抗血清效价为1/214,试验结果显示,此抗血清能用于葡萄植株样品中GLRaV-3不同分离物检测。
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| 关 键 词： | 葡萄融合蛋白 ACP-ELISA Western blot 分离物 |
| 收稿时间： | 2012/4/28 0:00:00 |
Preparation and application of antiserum against Grapevine leafroll associated virus 3 |
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| WANG Ke-shuang,FEI Fei,LV Mu-di,LI Jie and CHENG Yu-qin.Preparation and application of antiserum against Grapevine leafroll associated virus 3[J].Journal of China Agricultural University,2012,17(4):81-85. |
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| Authors: | WANG Ke-shuang FEI Fei LV Mu-di LI Jie and CHENG Yu-qin |
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| Institution: | *(Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, Key Laboratory of Beijing Municipality/College of Agronomy and Biotechnology, China Agricultural University,Beijing 100193,China) |
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| Abstract: | The pMD18-CP containing CP gene of Grapevine leafroll associated virus 3(GLRaV-3) was used for PCR amplification in order to obtain a specific antiserum for reliable GLRaV-3 diagnosis in routine tests.The PCR products of GLRaV-3 CP gene were cloned into the expression vector pET-28a.The resulted recombinant plasmid pET-G3CP was transformed into E.coli BL21(DE3).SDS-PAGE analysis showed that the recombinant GLRaV-3 CP was expressed as a 40 ku inclusion body protein at high level induced by IPTG at final concentration of 1 mmol/L.Antiserum was prepared after the rabbit was immunized with the purified recombinant CP.The antiserum titer was determined to be 1/214,and the antiserum was specific and sensitive for different GLRaV-3 isolates detection. |
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| Keywords: | grape recombinant protein ACP-ELISA Western blot isolate |
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