摘要
利用PCR方法分别扩增番木瓜环斑病毒(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)的外壳蛋白(coat protein,CP)基因,并连接到原核表达载体p ET-28a上,获得重组质粒p ET28-PRSV-CP、p ET28-PLDMV-CP和p ET28-Pa MV-CP,通过转化、诱导表达后,经SDS-PAGE分析显示,这3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以可溶蛋白形式存在。利用Ni2+-NTA琼脂糖亲和层析纯化获得了3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3种抗血清与对应的诱导表达蛋白发生特异反应。再采用抗血清建立一种快捷、简便和低成本的ID-ELISA(Indirect enzymelinked immunosorbent assay)技术,并利用此技术对海南岛283个疑似感病的番木瓜样品进行检测鉴别,初步掌握了海南岛3种主要番木瓜病毒的分布及感病情况,为下一步该病的有效防治策略提供科学依据。
The CP genes were amplified from 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 the 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 antiserum preparation. 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 were tested by ID-ELISA. This study would preliminarily establish the distribution of papaya virus in Hainan, and provid scientific basis and a effective method for the prevention of papaya virus.
出处
《热带作物学报》
CSCD
北大核心
2015年第10期1866-1872,共7页
Chinese Journal of Tropical Crops
基金
国家自然科学基金(No.31171822
No.31301639
No.31371918)
作者简介
杨勇（1987-），男，实验员；研究方向：分子生物学；E-mail：yybiology@163．com。
通讯作者（Corresponding author）：周鹏（ZHOUPeng），E-mail：zhp6301@126．com；
王锐萍（WANG Ruiping），E-mail：wrp@hainnu．edu．cn。
