草莓轻型黄边病毒CP蛋白的原核表达及抗血清制备  

Prokaryotic expression and antiserum preparation of CP protein of strawberry mild yellow edge virus

作　　者：卢静 韩金成 孙遗鑫 蒋磊[1,2,3] 江彤 LU Jing;HAN Jincheng;SUN Yixin;JIANG Lei;JIANG Tong(School of Plant Protection,Anhui Agricultural University,Hefei 230036,China;Anhui Province Key Laboratory of Integrated Pest Management on Crops,Hefei 230036,China;Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes,Hefei 230036,China)

机构地区：[1]安徽农业大学植物保护学院,合肥230036 [2]作物有害生物综合治理安徽省重点实验室,合肥230036 [3]植物病虫害生物学与绿色防控安徽普通高校重点实验室,合肥230036

出　　处：《植物病理学报》2023年第5期863-869,共7页Acta Phytopathologica Sinica

基　　金：安徽省重点研发项目(202004a06020013);安徽省高等学校科学研究项目(2022AH050920);国家自然科学基金项目(32072386)。

摘　　要：利用PCR技术扩增草莓轻型黄边病毒(strawberry mild yellow edge virus, SMYEV)中国分离物的外壳蛋白(coat protein, CP)基因,将SMYEV CP基因克隆至原核表达载体pET-32a,重组质粒pET-cp转化大肠杆菌BL21菌株,37℃振荡培养,1 mmol·L^(-1)IPTG诱导表达,检测发现CP融合蛋白大量存在于细胞裂解上清液中。Ni^(2+)-NTA亲和树脂纯化CP融合蛋白,Western blot可检测到较单一的特异性条带,分子量约为46 kDa。用纯化的CP融合蛋白免疫家兔获得多抗血清,间接ELISA法测定抗血清效价达1∶512 000。以此多抗血清建立了Dot-ELISA和胶体金试纸条检测方法,不仅可以检测感染SMYEV的草莓样本,用于感病草莓的早期诊断和鉴定,还可以用于SMYEV CP蛋白的瞬时表达和系统表达等分析。The coat protein(CP) gene of the Chinese isolate of strawberry mild yellow edge virus(SMYEV) was amplified by PCR. The gene was cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid pET-cp was transformed into Escherichia coli strain BL21, and then incubated at 37 °C with 1 mmol·L^(-1) IPTG to express the target protein. The result showed that fusion protein CP was present abundantly in the cell lysate supernatant. The fusion protein CP was purified by Ni^(2+)-NTA affinity resin, and western blot exhibited a single specific band with an approximate molecular weight of 46 kDa. The purified fusion protein CP was used to immunize rabbits and obtain polyclonal antibody. Indirect ELISA detection showed that the titer of antiserum to fusion protein reached 1: 512 000. Dot-ELISA and colloidal gold strip assays were established with the polyclonal antibody. These methods can be used not only for early diagnosis of SMYEV-infected strawberry samples, but also for the analyses of transient local and systemic expression of SMYEV CP protein.

关 键 词：草莓轻型黄边病毒 外壳蛋白 原核表达 抗血清 

分 类 号：S432.41[农业科学—植物病理学]