甘薯潜隐病毒莲藕分离物辅助成分-蛋白酶基因原核表达及抗血清制备  

Prokaryotic expression of sweet potato latent virus-lotus helper-component proteinase and antiserum preparation

作　　者：王凌琪 董婷婷[1] 陈雯[1] 甘海锋 徐小伟 秦朗 李良俊[1] 贺振[1] WANG Lingqi;DONG Tingting;CHEN Wen;GAN Haifeng;XU Xiaowei;QIN Lang;LI Liangjun;HE Zhen(School of Horticulture and Plant Protection,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学园艺与植物保护学院,扬州225009

出　　处：《植物保护》2022年第6期319-325,共7页Plant Protection

基　　金：江苏省自然科学基金(BK20211323);中央本级重大增减支项目(2060302);国家特色蔬菜产业技术体系(CARS-24-B-03)。

摘　　要：根据已报道的甘薯潜隐病毒莲藕分离物sweet potato latent virus-lotus(SPLV-lotus)核苷酸序列设计引物,采用RT-PCR从感病莲藕样品中扩增获得SPLV-lotus辅助成分-蛋白酶基因(HC-Pro),大小为1375 bp。通过序列测定和分析后,将其克隆到原核表达载体pGEX4T-1,转化大肠杆菌Rosetta(DE3),经IPTG诱导并纯化获得大小为74 kD的融合蛋白pGEX4T-1-HC-Pro^(SPLV-lotus)。SDS-PAGE结果显示,融合蛋白获得过量表达。以纯化蛋白为抗原免疫新西兰大白兔制备抗血清,采用ELISA和Western blot对抗血清效价和特异性进行检测,当HC-Pro抗血清稀释比为1∶256000时,ELISA显色后OD_(450)读数仍高于0.6,表明所制备抗血清合格;Western blot结果显示,在感染SPLV-lotus植株样品中仍能检测到单一目的条带。这些结果表明,制备的抗血清效价合格且可用于SPLV-lotus的特异性检测。In this study,the helper-component proteinase gene(HC-Pro,1375 bp)of sweet potato latent virus-lotus(SPLV-lotus)was amplified from lotus by RT-PCR using primers designed according to previously reported SPLV-lotus sequences.After sequencing and analysis,the HC-Pro gene was fused with an expression vector pGEX4 T-1 to produce the recombinant construct pGEX4 T-1-HC-Pro^(SPLV-lotus),which was then transformed into Escherichia coli Rosetta(DE3).The recombinant protein was induced with IPTG.SDS-PAGE analysis showed that the HC-Pro protein(approximately 74 kD)was expressed at a high level.The purified HC-Pro protein was used as antigen for raising antiserum in rabbits.Titer of the antiserum was measured by ELISA,and its specificity was tested by Western blot.The results showed that,when the dilution ratio of HC-Pro antiserum was 1∶256000,the OD_(450) reading remained higher than 0.6,indicating that the prepared antiserum was qualified for the development of serological detection.Western blot revealed a specific band in samples infected with SPLV-lotus,while no reaction was detected in healthy plants.These results suggested that the antiserum could be used for specific detection of SPLV-lotus.

关 键 词：甘薯潜隐病毒莲藕分离物 辅助成分-蛋白酶 原核表达 抗血清 

分 类 号：S436.32[农业科学—农业昆虫与害虫防治]