重组甘蔗花叶病毒E株系外壳蛋白的纯化及其多克隆抗体制备

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。

Purification of Recombinant Coat Protein of Sugarcane Mosaic Virus Strain E and Preparation of its Polyclonal Antibody

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.