侵染辣椒的黄瓜花叶病毒CP基因的序列分析、原核表达及抗血清制备  

Sequence Analysis,Prokaryotic Expression of CP Gene of Cucumber Mosaic Virus Infecting Pepper and Antiserum Preparation

作　　者：刘金亮[1] 王凤婷[1] 侯春喜[2] 魏毅[1] 张世宏[1] 潘洪玉[1] 

机构地区：[1]吉林大学植物科学学院,长春130062 [2]吉林大学超分子材料与结构国家重点实验室,长春130012

出　　处：《生物技术通报》2010年第8期110-115,共6页Biotechnology Bulletin

基　　金："十一五"国家科技支撑计划项目(2006BAD08A08);吉林大学博士科研启动基金项目(4305050102B2);吉林大学基本科研业务费项目(200903376)

摘　　要：从吉林长春感病辣椒上获得一黄瓜花叶病毒(Cucumber mosaic virus,CMV)分离物(CMV-CC),根据GenBank中已登录的CMV外壳蛋白(coat protein,CP)基因核苷酸序列设计简并引物,通过RT-PCR的方法克隆到了长度为657 bp的目的片段。序列分析表明,CMV-CC与CMVI组各分离物核苷酸同源性为93.2%-97.9%。根据完整CP基因核苷酸序列构建的系统进化树显示:38个CMV分离物可分为3个组,CMV-CC属于CMV的IB亚组。将CMV-CC CP基因与原核表达载体pET-22b(+)连接,在大肠杆菌BL21(DE3)诱导表达出分子量约27 kD的融合蛋白。表达的融合蛋白经树脂纯化后免疫家兔制备了抗血清。用间接ELISA测定抗血清效价为1/4 096。Western blotting分析表明制备的抗血清对CP有高度特异性,为准确、快速地检测CMV奠定了基础。One isolate of Cucumber mosaic virus,CMV-CC,was obtained from infected pepper plants in Changchun,Jilin province.The 657 bp coat protein（CP） gene of CMV-CC was amplified by RT-PCR.The obtained CP gene sequences were compared with sequences of CMV that are available in the GenBank.Results showed that the CP gene of CMV-CC shared similarity of 93.2%-97.9% with those of other CMV isolates in group I at nucleotide acid level.The phylogenetic tree constructed with the complete nucleotide sequence of the CP genes showed that 38 CMV isolates were divided into 3 groups,and CMV-CC belonged to subgroup IB.The CP gene of CMV-CC was inserted into expression vector pET-22b（＋） and transferred into E.coli BL21（DE3）.SDS-PAGE showed that CMV-CC CP gene was expressed as a 27 kD fusion protein when induced with IPTG.High specific antiserum against to CMV was prepared after the rabbit was immunized with the purified recombinant protein.The titer was 1： 4096 in indirect ELISA test.Result of Western blotting showed that the antiserum obtained could specifically react with CMV CP.

关 键 词：黄瓜花叶病毒 辣椒 CP基因 序列分析 原核表达 抗血清制备 

分 类 号：S436.418[农业科学—农业昆虫与害虫防治]