摘要
【目的】制备针对南方根结线虫食道腺蛋白MiPDCD6多克隆抗体,为进一步研究南方根结线虫MiPDCD6蛋白的致病机制提供技术支持和材料准备。【方法】将扩增MiPDCD6基因的功能片段,与原核表达质粒pET-32a(+)构建重组质粒pET-32a-MiPDCD6,然后将重组质粒转化大肠杆菌BL21细胞进行诱导表达;将纯化的根结线虫Mi PDCD6融合蛋白免疫雄性新西兰大白兔,获得高效价高纯度的多克隆抗体。【结果】构建的重组质粒pET-32a-MiPDCD6转化大肠杆菌BL21细胞后,在诱导剂IPTG浓度1.0 mmol·L^(-1)、摇床温度37℃、转速150r·min^(-1)和振荡培养5 h条件下,成功表达MiPDCD6融合蛋白。ELISA和SDS-PAGE检测表明:将纯化的根结线虫Mi PDCD6融合蛋白免疫雄性新西兰大白兔,获得高效价高纯度的多克隆抗体,效价约为1∶50 000。【结论】明确了MiPDCD6基因原核表达条件;制备获得的根结线虫MiPDCD6蛋白的多克隆抗体效价和纯度较高,可用于后续研究MiPDCD6蛋白在南方根结线虫致病机理中发挥的功能。
【Objective】The polyclonal antibody against MiPDCD6 protein in the esophageal glands of Meloidogyne incognita was prepared to study the pathogenic mechanism of the root-knot disease on plants transmitted by the nematode.【Method】The functional fragment of MiPDCD6 was amplified by PCR to construct recombinant plasmid pET-32a-MiPDCD6 and transform it into Escherichia coli BL21 cells for MiPDCD6 fusion protein induction and expression.Polyclonal antibodies were prepared by immunizing male New Zealand white rabbits with purified MiPDCD6 expression protein.Titer and purification of the obtained antibody were verified using ELISA and SDS-PAGE techniques.【Result】Under IPTG concentration of 1.0 mmol·L^(-1) at 37℃with constant rotation of 150 r·min^(-1) for 5 h,MiPDCD6 transformed in the E.coli BL21 was clearly expressed.The secured polyclonal antibody was highly specific with a high titer of approximately 1∶50000 as shown by ELISA and SDS-PAGE.【Conclusion】The prokaryotic expression conditions of MiPDCD6 were determined.The polyclonal antibody obtained had a high titer and specification against MiPDCD6 and was considered adequate for studying the pathogenesis of the root knot disease on plants infected through M.incognita.
作者
陈晨
高永峰
王新荣
袁永强
蔡书静
刘松松
叶雯华
王燕
CHEN Chen;GAO Yongfeng;WANG Xinrong;YUAN Yongqiang;CAI Shujing;LIU Songsong;YE Wenhua;WANG Yan(Guangdong Province Key Laboratory of Microbial Signals and Disease Control,College of Plant Protection,South China Agricultural University,Guangzhou,Guangdong 510642,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2023年第3期322-328,共7页
Fujian Journal of Agricultural Sciences
基金
广东省基础与应用基础研究基金(2019A1515012080)
国家自然科学基金项目(31171825、30771409)。
