【摘要】 本研究利用PCR技术扩增西瓜花叶病毒（watermelon mosaic virus, WMV）安徽分离物的6k2基因，将该基因克隆至原核表达载体pET-32a,获得重组质粒pET-6k2,转化大肠杆菌BL21菌株，经1 mmol/L IPTG 37℃4 h诱导表达，Ni²⁺-NTA亲和柱纯化融合蛋白，Western blot检测到的特异性条带分子量约为26 kD。以6k2融合蛋白作为抗原，免疫家兔获得多抗血清，间接ELISA法测定抗血清效价达1∶218 000以上。6k2多抗血清可用于检测感染WMV的瓜类样本，有助于感病瓜类作物的早期诊断，还可用于检测本氏烟中瞬时表达的6k2蛋白，为研究6k2蛋白功能奠定了基础。更多还原

【Abstract】 In this study, the 6k2 gene of a watermelon mosaic virus（WMV） isolate from Anhui province was amplified by polymerase chain reaction（PCR） and cloned into the prokaryotic expression vector pET-32a to generate the recombinant plasmid pET-6k2. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with 1 mmol/L isopropyl β-D-1-thiogalactopyranoside（IPTG） at 37℃ for 4 h. The fusion protein was purified using Ni²⁺-NTA affinity chromatography, and a specific band with an approximate molecular weight of 26 kD was detected by Western blot analysis. The purified 6k2 fusion protein was used as an antigen to immunize rabbits for the production of polyclonal antiserum. The antiserum titer, determined by indirect enzyme-linked immunosorbent assay（ELISA）, exceeded 1∶218 000. The 6k2 polyclonal antiserum was effective in detecting WMV-infected cucurbit samples, facilitating early diagnosis of virus-infected cucurbit crops. In addition, it was successfully applied to detect transient expression of the 6k2 protein in Nicotiana benthamiana, thereby providing a useful tool for further functional studies of the 6k2 protein.更多还原