玫瑰叶畸形病毒外壳蛋白基因原核表达与抗血清制备
Prokaryotic expression and antiserum preparation of coat protein of rosa rugosa leaf distortion virus
张秀琪 1聂张尧 1李梦林 1苏江月 1丁泽慧 1张宗英 1韩成贵 1王颖1
作者信息
- 1. 中国农业大学植物保护学院,农业农村部作物有害生物监测与绿色防控重点实验室,北京 100193
- 折叠
摘要
玫瑰叶畸形病毒(rosa rugosa leaf distortion virus,RrLDV)属于番茄丛矮病毒科Tombusviridae天竺葵环斑病毒属Pelarspovirus,能够引起月季叶片畸形、黄化以及提前衰老等症状,是危害月季的病毒之一.为建立玫瑰叶畸形病毒的快速检测方法,采用热硼酸盐法提取发病月季叶片总RNA,通过RT-PCR扩增该病毒外壳蛋白基因,连接至原核表达载体pDB-His-MBP上,导入大肠杆菌Escherichia coli BL21以IPTG,诱导蛋白表达,获得浓度为8 mg/mL的重组蛋白,制备了兔多抗血清.Western blot检测血清效价为1∶2 048 000,当效价为1∶1 000时,灵敏度为1∶1 024.血清学检测结果与RT-PCR检测结果一致,表明该抗血清可用于RrLDV的检测,有利于检测该病毒的发生和分布.
Abstract
Rosa rugosa leaf distortion virus(RrLDV)belongs to the genus Pelarspovirus in the family Tombusviridae,which can infect roses and cause symptoms such as leaf deformity,yellowing and premature aging.In order to establish a rapid detection method for detecting RrLDV,total RNA of susceptible rose leaves was extracted by hot borate method.The gene which encodes coat protein of the virus was amplified by RT-PCR and constructed to the prokaryotic expression vector pDB-His-MBP.The vector was transformed into Escherichia coli BL21 and the expression of the recombinant protein was induced by IPTG.The recombinant protein with a concentration of 8 mg/mL was obtained and the polyclonal anti-rabbit antiserum was produced.Western blotting showed the titer of the antiserum was 1∶2 048 000 and its sensitivity was 1∶1 024 when the antiserum was diluted to 1∶1 000.The serological detection result was consistent with RT-PCR,indicating that the antiserum can be used to detect the occurrence and distribution of RrLDV.
关键词
玫瑰叶畸形病毒/外壳蛋白/原核表达/抗血清制备Key words
rosa rugosa leaf distortion virus/coat protein/prokaryotic expression/antiserum preparation引用本文复制引用
基金项目
国家级大学生创新训练项目(201910019028)
出版年
2024
NETL
NSTL
万方数据