【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789 bp。将TEV CP基因克隆到pM D 18-T S im p le V ector,经E coRⅠ/S acⅠ双酶切得到目的片段,并将其定向插入到pET 30a载体中构建了原核表达载体pET 30-TEV CP,重组质粒经E coRⅠ和S acⅠ双酶切鉴定及基因测序验证正确后,转化大肠杆菌BL 21,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37 ku。以表达产物为抗原免疫家兔,制备了特异性抗血清,其效价为1/1 204。W estern b lot检测结果表明,制备的抗血清可用于检测田间的发病植株。更多还原

【Abstract】 Coat protein(CP) gene of tobacco etch virus was cloned into pMD18-T simple vector by means of RT-PCR amplification,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/SacⅠ and 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.更多还原