甘蔗宿根矮化病多克隆抗体和免疫磁珠的制备

Preparation of Polyclonal Antibody Against Ratoon Stunting Disease and Its Immunomagnetic Beads

本研究通过制备Lxx(Leifsonia xyli subsp.xyli)免疫磁珠,实现对甘蔗Lxx进行分离,用于病原菌与甘蔗互作的深入研究。以RSD PCR检测呈阳性的感病甘蔗为材料,分离培养Lxx抗原进行多克隆抗体制备,利用磁性Fe3O4纳米粒子与Lxx多克隆抗体偶联制备免疫磁珠。根据菌落的形态特征,并结合PCR验证确认所分离到的菌是甘蔗宿根矮化病的致病菌Lxx,免疫兔子后获得了较好的多克隆抗体,间接ELISA测定Lxx多克隆抗体的效价高于204 800。免疫磁珠外观成球性较好,粒径大小在125-268 nm之间,对菌培养液中Lxx亲和性强分离物可用于进一步的PCR实验。Lxx免疫磁珠的成功制备,能够快速实现Lxx的分离与富集,为Lxx与甘蔗互作时期下甘蔗感病性检测和病原菌的富集提供了便捷高效的技术手段。

This study is to isolate sugarcane Lxx by preparing Lxx immunomagnetic beads（IMB）for the further study the interaction between pathogen and sugarcane. The pathogen of Lxx was isolated from the RSD PCR-verified infected sugarcane for the preparation of polyclonal antibody. Then the Lxx polyclonal antibody was coupled with magnetic Fe3 O4 nano particle for preparing IMB. The isolates were identified as Lxx according to the morphological characteristics of colony and the PCR results. The fine polyclonal antibody was obtained by immunizing rabbits with Lxx. The titer of the polyclonal antibody against Lxx was greater than 204,800 by indirect ELISA. The appearance of the IMB was globular,and the particulates were 125-268 nm in diameter. The isolates with high affinity for Lxx in the culture media can be used for PCR reaction. The successful preparation of Lxx IMB can rapidly isolate and enrich Lxx,which provides a convenient and efficient technique for the detection of pathogenic bacteria under the interaction of Lxx and sugarcane.