苹果茎沟病毒外壳蛋白基因的原核表达及抗血清的制备  

Prokaryotic expression of cp genes from apple stem grooving virus and production of antisera against recombinant coat proteins

作　　者：宋艳苏[1] 郑银英[2] 李丽娜[1] 王国平[1] 洪霓[1] 

机构地区：[1]华中农业大学植物科学技术学院湖北省作物病害监测与安全控制重点实验室,武汉430070 [2]石河子大学生命科学学院农业生物技术重点实验室,新疆石河子832003

出　　处：《果树学报》2010年第5期752-756,共5页Journal of Fruit Science

基　　金：国家梨产业技术体系(nycytx-29-08)

摘　　要：构建了来源于梨的苹果茎沟病毒(Apple stem grooving virus,ASGV)分离物P-4-1-69和P-L2的原核表达载体pET-P-4-1-69和pET-P-L2,转化大肠杆菌BL21(DE3),在1 mmol.L-1 IPTG下诱导表达。SDS-PAGE和用商业化ASGV抗体对诱导表达产物进行Western Blot分析结果表明,2个分离物的外壳蛋白基因(cp基因)在大肠杆菌中成功地进行了高效表达,重组外壳蛋白的大小约为31 ku。分别用含分离物P-4-1-69和P-L2的重组外壳蛋白的胶条免疫大耳白兔,制备了抗ASGV重组外壳蛋白的抗血清。采用间接ELISA法用所制备的抗血清对回收的重组外壳蛋白进行检测,当抗分离物P-4-1-69和P-L2的重组外壳蛋白的抗血清分别稀释512 000倍和64 000倍时,检测结果仍为阳性。Western Blot分析和组织免疫印迹检测(Tissue blotting immunoassay,TBIA)结果表明,纯化的抗体具有较强的特异性,可有效检测梨样品中的ASGV。In this study, cp genes of two isolates of Apple stem grooving virus from pear trees, P-4-1-69 and P-L2, were constructed into the bacterial expression vector pET-28a（＋）. The recombinant plasmids pET-P-4-1-69 and pET-P-L2 con- taining cp genes were obtained and transformed into Escherichia coli （E. coli） strain BL21 （DE3）. SDS-PAGE and Western Blot analysis with alkaline phosphatase-conjugated ASGV IgG indicated that cp genes of two ASGV isolates were expressed effectively and molecular weight of these recombinant coat proteins was approximately 31 ku. These recombinant coat proteins were used to raise antisera in rabbits. In Indirect-ELISA, antisera against recombinant coat protein of isolate P-4-1-69 diluted 1：512 000 or isolate P-L2 diluted 1：64 000 reacted strongly with homologous proteins expressed in E. coli. These purified antibodies showed to possess high specificity to their recombinant coat proteins in Western Blot and native virus coat proteins in infected pear shoot in vitro by tissue blotting immunoassay （TBIA）.

关 键 词：苹果茎沟病毒 CP基因 原核表达 抗血清 

分 类 号：S661.2[农业科学—果树学]