李属坏死环斑病毒怀柔分离物CP基因的克隆、原核表达及其抗血清制备  

Cloning of the CP gene,prokaryotic expression and antiserum preparation of Prunus necrotic ringspot virus(PNRSV-Hr)

作　　者：李明福 陈洪俊 陈定虎 邱德义 杨雷亮 赵世恒 

机构地区：[1]中国检验检疫科学院,北京100029 [2]广东中山出入境检验检疫局技术中心,528400

出　　处：《植物保护》2009年第4期29-33,共5页Plant Protection

基　　金：国家自然科学基金项目(30270892)

摘　　要：本研究通过RT-PCR技术获得了包含李属坏死环斑病毒怀柔分离物CP蛋白基因的DNA片段,构建了针对pET29a载体的两种表达质粒;转化大肠杆菌BL21(DE3),并经IPTG诱导表达了带不同融合肽段的PNRSV1(25 ku)和PNRSV2(29 ku)融合蛋白。经过Ni-NTA亲和柱与SDS-PAGE分离纯化,获得大量表达融合蛋白,并免疫家兔制备了融合表达蛋白的特异性抗体。间接ELISA测定其效价分别为8×103和3.2×104。The Prunus necrotic ringspot virus （PNRSV-Hr） coat protein（CP）gene was amplified by RT-PCR with the primers of PNRSV CP gene and then the CP gene was inserted into two different sites of the prokaryotic expression vector pET29a and transformed into E. coli BL21 （DE3）. The results of SDS-PAGE showed that PNRSV CP gene was expressed effectively when induced with IPTG, as a 25 ku PNRSV-6His （PNRSV1） fusion protein or a 29 ku S-tag-PNRSV-6His （PNRSV2） fusion protein. The antisera of high specificity was obtained when the rabbits were immunized with the purified recombinant proteins and the titers of the two antisera were determined to be 8×10^3 and 3.2×10^4 by indirect ELISA, respectively.

关 键 词：李属坏死环斑病毒 外壳蛋白 原核表达 蛋白纯化 抗体 

分 类 号：S436.629[农业科学—农业昆虫与害虫防治]