【作者】 杨勇； 庹德财； 沈文涛； 言普； 黎小瑛； 周鹏； 王锐萍；

【Author】 YANG Yong;TUO Decai;SHEN Wentao;YAN Pu;LI Xiaoying;ZHOU Peng;Wang Ruiping;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences;College of Life Science,Hainan Normal University;

【机构】 中国热带农业科学院热带生物技术研究所； 海南师范大学生命科学学院；

【摘要】 利用PCR方法从本实验室保存的番木瓜环斑病毒(papaya ringspot virus,PRSV)、番木瓜畸形花叶病毒(papaya leaf distortion mosaic virus,PLDMV)和番木瓜花叶病(papaya mosaic virus,Pa MV)的全长基因组的质粒上分别扩增了三种病毒的外壳蛋白(coat protein,CP)基因,并连接到原核表达载体p ET-28a上,将获得的重组质粒p ET28-PRSV CP、p ET28-PLDMV CP和p ET28-Pa MV CP分别转化到大肠BL21(DE3),IPTG诱导后,SDS-PAGE分析显示这三种CP蛋白在大肠杆菌中高效表达,其中PRSV CP蛋白和PLDMV CP以包涵体形式存在,Pa MV CP以融合蛋白形式存在。利用Ni+-NTA琼脂糖亲和层析获取了三种病毒的重组CP蛋白,并免疫大白兔获得高效价的抗体。Western blot检测结果表明,这三种抗血清与对应的诱导表达蛋白发生特异反应。采用上述抗血清建立了一种快捷、简便和低成本的ID-ELISA技术(Indirect enzyme-linked immunosorbent assay),并利用此技术对海南岛283个疑似感病番木瓜样品进行了检测鉴别,初步掌握了海南岛三种主要番木瓜病毒的分布及感病情况,这为下一步采取针对性的有效防治策略提供了科学依据。更多还原

【Abstract】 The CP genes were amplified from the PRSV,PLDMV and Pa MV sequences by PCR,which were kept in our laboratory.Then the CP genes were connected to prokaryotic expression vector of p ET-28 a,after identification by enzyme digestion and sequencing,the three recombinant plasmids of p ET28-PRSV CP,p ET28-PLDMV CP and p ET28-Pa MV CP were transformed into expressing bacteria BL21(DE3).The CP genes were expressed efficiently with IPTG inducing by SDS-PAGE analysis.PRSV CP and PLDMV CP existed in the form of inclusion body,while Pa MV CP expressed in the soluble form.The expressed proteins were purified with Ni2+-NTA agarose affinity chromatography and were used as antigens to immunize the rabbits for antiserumpreparation.Western blot analysis confirmed that the antiserum only reacted with the corresponding expressed induced protein.283 of unhealthy papaya leaves collected on the whole Hainan islands were tested by ID-ELISA,this study has preliminary established the distribution of papaya virus in Hainan,and provided scientific basis and a effective method for the prevention of papaya virus.更多还原