大麦条纹花叶病毒中国株CP基因克隆分析、原核表达及抗血清制备  

Molecular cloning,sequencing and expression of CP gene of Barley stripe mosaic virus China strain(BSMV-CH) in Escherichia coli BL21 and its antiserum preparation

作　　者：孙现超[1] 安德荣[1] 薛杨[1] 

机构地区：[1]西北农林科技大学植物保护学院,杨凌712100

出　　处：《植物病理学报》2006年第5期397-403,共7页Acta Phytopathologica Sinica

基　　金：西北农林科技大学"创新团队建设计划"项目资助

摘　　要：首次用PCR方法，自大麦条纹花叶病毒中国株（BSMV-CH）的RNA2（GenBank登录号为：AY789694，未报道）中扩增出外壳蛋白（coat protein，CP）基因，并亚克隆到pMD18-T载体上进行测序分析。结果表明BSMV—CH的CP全长597bp，它与BSMV其它株系CP的核苷酸同源性和氨基酸同源性分别为93．1％～93．8％和91．4％-92．4％；与同属的早熟禾半潜病毒（PSLV）和剪秋罗环斑病毒（LRSV）的核苷酸同源性分别为53．0％和41．8％，氨基酸同源性分别为48．1％和40．0％。根据大麦病毒属病毒已经报道的CP氨基酸序列绘制系统发育树，分析表明分离的各毒株在进化上存在明显地理位置的相关性。把CP基因亚克隆到GST融合表达载体pEGX-6p-1上，优化IFFG诱导终浓度后，在37℃ IFFG终浓度为1．0mmol/L时，融合蛋白GST-CP在大肠杆菌BL21中得到了特异表达。12％SDS—PAGE表明，表达产物大小为48．5kDa，主要以包含体形式存在。对蛋白进行定量后，用冰冷的KCl显色，分离表达的蛋白质特异条带制备抗原，免疫家兔得到抗血清，酶联法（enzyme-linked immunosorbant assay，ELISA）测定的抗血清效价为1／6400。用抗血清对感病大麦和健康大麦进行检测，结果表明抗血清有很高特异性。The coat protein （CP） was amplified from RNA2 （GenBank accession number AY789694 ） of Barley stripe mosaic virus Chain strain （BSMV-CH） by PCR, and subcloned into pMD18-T vector for sequence analysis for the first time in China. Its length was 597 bp. Compared with the other stains of BSMV, the identifies of nucleofides were between 93.1% and 93.8% ; the identifies of amino acids were between 91.4% and 92.4%. Compared with Poa semilatent virus （PSLV） and Lychnis ringspot virus （LRSV）, the identifies of nucleotides were 53.0% and 41.8% and that of amino acids were 48.1% and 40.0%, respectively. Phylogenetic tree analysis showed that there was correlation between BSMV isolates and geographic regions. The coat protein was subcloned to pEGX-6p-1, an expression vector fused with GST. The fusion protein was successfully expressed in E. coil BL21 induced with 1.0 mmol/L IPTG at 37℃. 12% SDS-PAGE showed that the expression product with molecular weight 48.5 kDa was mainly in the form of inclusion body. Rabbit was immunized with the expressed fusion protein as antigen and antiserum was obtained. The fiter of the antiserum was determined to be 1/6 400 by enzyme-linked immunosorbant assay. The antiserum had high specificity to barley leaves infected by Barley stripe mosaic virus.

关 键 词：大麦条纹花叶病毒 外壳蛋白 系统发育树 融合蛋白 抗血清 

分 类 号：S432.41[农业科学—植物病理学]