摘要
目的:制备可用于甘蔗花叶病毒(ScMV)E株系(ScMV-E)检测用多克隆抗体。方法:将ScMV-E外壳蛋白(CP)基因连接到pET29a(+)上,经PCR检测、酶切及测序鉴定获得重组质粒pET29a-CP,在大肠杆菌BL21(DE3)中诱导表达重组ScMV-E外壳蛋白;采用His Trap Kit纯化目的蛋白,作为抗原免疫新西兰大白兔,制备特异性抗体;通过间接ELISA、Western blot和组织印迹法检测所制备抗体的特异性。结果:SDS-PAGE分析表明,重组融合蛋白含6个组氨酸标记,相对分子质量约43000;Western blot检测显示所获得的抗体特异性良好,间接ELISA法测得血清的效价为1:81 920;甘蔗叶片的组织印迹检测结果显示杂交效果良好。结论:制备的多克隆抗体可直接用于ScMV-E检测,并有望用于制备ScMV-E检测试剂盒。
Objective： To prepare the polyclonal antibody detecting sugarcane mosaic virus strain E（ScMV-E）. Methods： The prokaryotic expression plasmid was constructed by ligating the target coat protein（CP） gene of ScMV-E into the vector pET29a（＋）. Through detection by PCR, digestion and sequencing, one of the positive clones was named as pET29a-CP and used for the following experiment. After SDS-PAGE analysis, the recombinant CP of ScMV-E purified by His Trap Kit was injected into rabbits, the corresponding polyclonal antibody specific to ScMV-E was obtained. Then SAP-ELISA, Western blot and tissue blot-ELISA was applied in the detection of its speciality. Results： The result of SDS-PAGE analysis showed that specific expression of a 43 kD fusion protein with 6 His trap was achieved. Western blot analysis confirmed the fact that the CP of ScMV-E was specifically expressed. Titer of ScMV-E antibody was 1：81 920 by SAP- ELISA. Tissue blot-ELISA analysis of sugarcane leaves collected in the field showed that the ScMV-E antibody can be used directly in the detection of ScMV-E. Conclusion： The polyclonal antibody obtained here could be used to detect ScMV-E directly or to prepare corresponding kit.
出处
《生物技术通讯》
CAS
2009年第1期69-71,102,共4页
Letters in Biotechnology
基金
国家高技术研究发展计划(2007AA100701)
福建省科技厅备案项目(F2007AA100701)
农业部"948"项目(2006-G37)
关键词
甘蔗花叶病毒
外壳蛋白
原核表达
纯化
多克隆抗体
组织印迹
sugarcane mosaic virus
coat protein
prokaryotic expression
purification
polyclonal antibody
tissue blot-ELISA
