百合无症病毒16kD基因的克隆、原核表达及抗血清制备  

Gene Clone,Prokaryotic Expression of Lily symptomless virus 16 kD Gene and Preparation of Its Polyclonal Antibody

作　　者：冀文婕 张郑瑶[2] 徐品三[1] 

机构地区：[1]大连理工大学生命科学与技术学院,辽宁大连116024 [2]大连理工大学生命与医药学院,辽宁盘锦124221

出　　处：《园艺学报》2016年第5期998-1004,共7页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31070621)

摘　　要：以感染百合无症病毒(LSV)的百合叶片为试材,克隆LSV16 k D基因,连接到原核表达载体p ET-28a(+)上。将获得的重组质粒p ET-28a(+)+16 k D转化大肠杆菌BL21(DE3),经IPTG诱导得到了高效表达的16 k D蛋白,融合蛋白分子量约为20 k D。融合蛋白经过镍柱纯化后作为抗原免疫注射小鼠,制备得到16 k D蛋白抗血清。Western blot分析显示所制备的抗血清与诱导表达的融合蛋白发生特异性反应;通过ELISA检测和RT-PCR检测百合样品,证实制备的抗血清与LSV侵染的百合叶片发生了相同的特异性反应。结果表明,目的蛋白表达成功,所制备的抗血清具有特异性,可用于LSV的快速检测、免疫组织化学以及16 k D蛋白功能研究。Unknown gene（16 kD）was amplified by RT-PCR from Lily leaves infected by Lily symptomless virus and cloned into prokaryotic expression vector p ET-28a（＋）. Then the recombinant plasmid vector ligated with His-tag and carried 16 kD gene was transformed into E. coli strain BL21（DE3）. Induced with IPTG,the protein was highly expression in E. coli and the molecular weight of the recombinant protein 16 kD was 20 kD. After purification with Ni-（2＋）-NTA affinity chromatography,polyclonal antibody 16 kD was raised in mouses. Western blot analysis showed that the antiserum reacted specially with 16 kD protein of LSV;ELISA and RT-PCR also confirmed that the antiserum reacted specially with lily leaves infected by LSV. Our results indicate that the interest protein expression was detected, the antiserum reacted specially with 16 kD protein and used for LSV rapid test,immunohistochemistry and functional study of 16 kD protein.

关 键 词：百合 百合无症病毒 16 k D 基因克隆 原核表达 抗血清制备 

分 类 号：S682.2[农业科学—观赏园艺] Q786[农业科学—园艺学]