| 徐小伟, 陈 雯, 丁诗文, 甘海锋, 张 坤, 贺 振*.甘蔗线条花叶病毒P1蛋白基因原核表达及抗血清制备[J].植物保护,2022,48(2):157-161. |
| 甘蔗线条花叶病毒P1蛋白基因原核表达及抗血清制备 |
| Prokaryotic expression of Sugarcane streak mosaic virus P1 protein gene and preparation of antiserum |
| 投稿时间：2021-01-20 修订日期：2021-04-07 |
| DOI：10.16688/j.zwbh.2021042 |
| 中文关键词: 甘蔗线条花叶病毒 P1蛋白 原核表达 抗血清 |
| 英文关键词:Sugarcane streak mosaic virus P1 protein prokaryotic expression antiserum |
| 基金项目:江苏省自然科学基金(BK20211323); 中央本级重大增减支项目(2060302); 扬州大学青蓝工程 |
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| 中文摘要: |
| 甘蔗线条花叶病毒Sugarcane streak mosaic virus(SCSMV)是引起甘蔗花叶病的主要病原之一, 在世界各大蔗区普遍发生, 严重威胁甘蔗产业的发展?建立快速有效的检测方法对于SCSMV的防控有着重要意义?本研究依据SCSMV P1基因序列合成一对引物, 扩增获得1 074 bp的目的基因, 将目的基因与原核表达载体pET28a连接, 获得pET28a-P1SCSMV?将连接正确的重组质粒转化大肠杆菌Rosetta菌株, 经IPTG诱导后, SDS-PAGE电泳检测显示在分子量约为42 kDa处有目的蛋白带, 与预期的SCSMV P1大小一致?融合蛋白主要是以可溶性蛋白的形式存在?利用镍柱亲和纯化重组的SCSMV P1蛋白, 并免疫健康新西兰大白兔, 制备兔抗血清?Western blot分析显示, 在对多个样品进行检测时制备的抗血清能特异性地识别SCSMV的P1蛋白, 在受SCSMV侵染的甘蔗植株中能检测到P1蛋白的表达, 而在健康植株中检测不到P1蛋白的表达?制备的抗血清稀释至1∶20 000时仍能特异地检测到目的蛋白条带, 说明通过大肠杆菌表达P1制备的SCSMV抗血清特异性强, 效价高?本研究为SCSMV的快速检测提供了有效方法? |
| 英文摘要: |
| Sugarcane streak mosaic virus (SCSMV) is one of the main pathogens that cause sugarcane mosaic disease. It occurs in major sugarcane-growing regions around the world and seriously threatens the development of the sugarcane industry. The establishment of rapid and effective detection methods to prevent and control SCSMV is of great significance. In this study, a pair of primers were synthesized based on the SCSMV P1 gene sequence, and the target gene with a size of 1 074 bp was amplified. The target gene was connected with the prokaryotic expression vector pET28a to obtain pET28a-P1SCSMV. The correct recombinant plasmid was transformed into Escherichia coli Rosetta strain. After induction by IPTG, SDS-PAGE electrophoresis showed that there was a band of the target protein at a molecular weight of about 42 kDa, which was consistent with the expected size of SCSMV P1. Fusion protein mainly existed in the form of soluble protein. The recombinant SCSMV P1 protein was obtained by affinity purification with a Ni2+-NTA column and healthy New Zealand white rabbits was immunized with recombinant SCSMV P1 protein to prepare rabbit antiseruM. Western blot analysis showed that the prepared antiserum could specifically recognize the SCSMV P1 protein when testing multiple samples. The expression of P1 protein could be detected in sugarcane plants infected by SCSMV, but not in healthy plants. When the prepared antiserum was diluted to 1∶20 000, the target protein band could still be detected specifically. This suggested that the SCSMV antiserum prepared by expressing P1 in E.coli had strong specificity and high titer, which provides favorable conditions for rapid detection of SCSMV. |
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