| 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 associated virus 3,GLRaV-3),本研究用含有GLRaV-3 CP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1 mmol/L 的IPTG进行诱导表达,SDS-PAGE 电泳分析显示,GLRaV-3 CP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。 |
| 关键词: 葡萄融合蛋白 ACP-ELISA Western blot 分离物 |
| DOI：10.11841/j.issn.1007-4333.2012.04.014 |
| 投稿时间：2012-04-28 |
| 基金项目:现代农业产业技术体系建设专项(CARS-30-bc-1) |
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| 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, CHENG Yu-qin
|
| (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) |
| 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. |
| Key words: grape recombinant protein ACP-ELISA Western blot isolate |
