| 张秀琪#, 聂张尧#, 李梦林, 苏江月, 丁泽慧, 张宗英, 韩成贵, 王 颖*.玫瑰叶畸形病毒外壳蛋白基因原核表达与抗血清制备[J].植物保护,2024,50(4):230-234. |
| 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 |
| 投稿时间：2023-06-24 修订日期：2023-11-01 |
| DOI：10.16688/j.zwbh.2023315 |
| 中文关键词: 玫瑰叶畸形病毒 外壳蛋白 原核表达 抗血清制备 |
| 英文关键词:rosa rugosa leaf distortion virus coat protein prokaryotic expression antiserum preparation |
| 基金项目:国家级大学生创新训练项目（201910019028） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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