| 摘要: |
| 根据烟草蚀纹病毒（TEV）CP序列设计合成上下游引物，利用RT-PCR方法获得了TEV CP基因，大小为789bp。将TEV CP基因克隆到pMD18-T Simple Vector，经EcoRⅠ/SacⅠ双酶切得到目的片段，并将其定向插入到pET30a载体中构建了原核表达载体pET30-TEV CP，重组质粒经EcoRⅠ和SacⅠ双酶切鉴定及基因测序验证正确后，转化大肠杆菌BL21，用IPTG进行诱导表达。结果表明，TEV CP基因在大肠杆菌中获得了高效表达，分子量约为37ku。以表达产物为抗原免疫家免，制备了特异性抗血清，其效价为1/1204。Western blot检测结果表明．制备的抗血清可用于检测田间的发病植株。 |
| 关键词: 烟草蚀纹病毒 外壳蛋白基因 原核表达 抗血清 |
| DOI： |
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| 基金项目:陕西省烟草专卖局攻关项目(2001KN-4);西北农林科技大学创新团队项目 |
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| Cloning of tobacco etch virus cp gene and prokaryotic expression in E.coli |
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| Abstract: |
| Coat protein (CP) gene of tobacco etch virus was cloned into pMD18-Tsimple vector by means of RT-PCRamplification,which were cleaved with restrict endonuclease EcoRⅠ/SacⅠ and inserted into pET30a.We constructed the expression vector of pET30-TEV CP which was cleaved with EcoRⅠ/SacⅠand gene sequenced exactly,and then transformed into BL21 host strain of E.coli.The target protein was induced by IPTG and the molecular size of expression product was about 37 ku.The product was used as antigen to immunize the rabbit and the titer of antiserum was 1∶1 204.Western blot showed that the preparated antiserum can be used to detect the tobaccos infected TEV. |
| Key words: tobacco etch virus coat protein prokaryotic expression antiserum |
