森林草莓RD21基因的克隆与蛋白酶域的原核表达及抗血清制备  

Cloning of Woodland Strawberry(Fragaria vesca)RD21 Gene,Prokaryotic Expression and Antiserum Preparation of Its Protease Domain

作　　者：余维琪 吴浩 杨先初 宋培培 江彤[1,3,4] YU Weiqi;WU Hao;YANG Xianchu;SONG Peipei;JIANG Tong(School of Plant Protection,Anhui Agricultural University,Hefei 230036,China;Pingdu Agriculture and Rural Affairs Bureau,Pingdu Shandong 266700,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]山东省平度市农业农村局,山东平度266700 [3]作物有害生物综合治理安徽省重点实验室,合肥230036 [4]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2300362

出　　处：《西北农业学报》2025年第4期702-708,共7页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：安徽省高等学校科学研究项目(2022AH050920);国家自然科学基金(32472518)。

摘　　要：为了明确森林草莓(Fragariavesca)RD21蛋白对草莓镶脉病毒致病性的影响,本研究采用原核表达技术获得RD21蛋白酶域(Protease)重组蛋白并制备其抗血清。先克隆森林草莓半胱氨酸蛋白酶RD21基因,分析RD21基因的遗传多样性。再将RD21基因蛋白酶域片段(RD21-3)克隆至原核表达载体pET-32a,重组质粒pET-RD21-3转化大肠杆菌,IPTG诱导RD21蛋白酶域融合蛋白表达。结果表明,RD21基因序列全长1410nt,编码470个氨基酸。序列比对发现,森林草莓RD21基因与9种蔷薇科植物RD21基因的序列相似性较高,为83.4%~94.2%。构建RD21基因系统关系树,发现森林草莓与其他蔷薇科植物的RD21基因聚成一个分支,说明森林草莓与蔷薇科植物的亲缘关系较近。用纯化的RD21蛋白酶域融合蛋白免疫家兔,获得融合蛋白的抗血清。ELISA法测定抗血清效价达1∶512000,Westernblot分析显示,制备的抗血清可与融合蛋白发生特异性反应,说明本研究制备的RD21蛋白酶域融合蛋白的抗血清能够成功检测出靶标蛋白。To elucidate the effect of the RD21 protein of woodland strawberry(Fragaria vesca)on the pathogenicity of strawberry vein banding virus,prokaryotic expression technology was used to produce the recombinant protein of RD21 protease domain,followed by the preparation of antiserum specific to the RD21 protease domain.First,the cysteine protease gene RD21 was cloned from F.vesca,and the genetic diversity of RD21 gene was analyzed.Subsequently,a fragment encoding the RD21 protease domain(RD21-3)was inserted into the prokaryotic expression vector pET-32a.The recombinant plasmid pET-RD21-3 was transformed into Escherichia coli,and IPTG induction was used to express the RD21 protease domain fusion protein.The results showed that the full-length sequence of RD21 gene comprises 1410 nt,encoding a protein consisting of 470 amino acids.Sequence alignment revealed that the RD21 gene of F.vesca exhibited a high sequence similarity(83.4%-94.2%)with corresponding genes from nine other Rosaceae species.Phylogenetic analysis revealed that RD21s genes from F.vesca clustered closely with those from other Rosaceae species,indicating a significant genetic relationship.The purified protein of RD21 protease domain was used to immunize rabbits,resulting in the production of polyclonal antibodies against the fusion protein.ELISA analysis indicated that the antiserum titer reached 1∶512000,and Western blot confirmed that the antiserum could react specifically with the fusion protein.These findings confirm that the target protein can be effectively detected using the RD21 protease domain antiserum.

关 键 词：森林草莓 半胱氨酸蛋白酶RD21基因 蛋白酶域 原核表达 抗血清 

分 类 号：S436.3[农业科学—农业昆虫与害虫防治]