侵染浙贝母的浙贝母病毒Y(FVY)CP基因的原核表达及抗血清制备(英文)  

Prokaryotic Expression of CP Gene of Fritillary virus Y Infecting Thunberg Fritillary and Antiserum Preparation

作　　者：韦传宝[1,2] 隗洋洋[2] 杨宇[2] 刘士亮[2] 胡皓雨[2] 何月[2] 

机构地区：[1]皖西学院安徽仿生传感与检测技术实验室,安徽六安237012 [2]皖西学院生物与制药工程学院,安徽六安237012

出　　处：《中药材》2011年第10期1498-1502,共5页Journal of Chinese Medicinal Materials

基　　金：安徽省教育厅自然科学基金重点项目资助(KJ2010A328;KJ2011A272)

摘　　要：目的:制备抗侵染浙贝母的浙贝母病毒Y(Fritillary virus Y,FVY)CP血清,为大田检测FVY和研究FVY与其他病毒之间的血清学关系奠定基础。方法:根据Genbank报道的FVY CP基因序列设计引物,扩增其CP基因。Blast分析FVY CP基因核苷酸序列与其他马铃薯Y病毒属成员CP基因的同源性。将CP基因插入表达载体pSBET,转化大肠杆菌BL21(DE3)Plys E菌株,通过IPTG诱导表达。经12%SDS-PAGE和5%～20%梯度SDS-PAGE两次纯化CP,免疫小鼠获得抗CP血清,采用Western blot分析抗体的特异性。用原核表达的17种马铃薯Y病毒属病毒CP蛋白作为抗原,检测抗FVY CP血清是否能与其进行交叉反应。采用ELISA分析抗体能否与天然FVY病毒离子结合。结果:FVY CP基因与油点草病毒Y(TrVY)CP基因、大豆花叶病毒半夏分离物CP基因和小西葫芦黄花叶病毒六安分离物CP基因分别有81.2%、68.1%和67.2%的同源性。制备的抗体对FVY CP具有高度特异性,同时能与大豆花叶病毒半夏分离物(SMV-P)CP蛋白有中等强度的结合,与小西葫芦黄花叶病毒CP蛋白有弱的结合。结论:抗体能够与天然FVY病毒离子结合,因此可以作为该病毒的大田检测。Objective ：To prepare antiserum against Fritillary virus Y （FVY） CP for detecting FVY and study serological relationships with other viruses. Methods： Specific primer was designed according to Genbank （accession：AM039800） to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pS- BET and expressed in Escherichia coli BI21 （ DE3 ） plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5%-20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis. Results： It shared 81.2% nucleotide acids identities with TrVY （Tricyrtis virus Y, AY 864850） CP gene, 68.1% with SMV-P （Soybean mosaic virus Pinellia strain, AJ507388. 2）CP gene and 67.2% with ZYMV（Zucchini yellow mosaic virus Luan isolate） CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P（Soybean mosaic virus Pinellia strain） and weakly to that of ZYMV（Zucchini yellow mosaic virus Luan isolate）. Conclusion：The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.

关 键 词：浙贝母 浙贝母病毒Y CP基因 原核表达 抗血清制备 

分 类 号：Q785[生物学—分子生物学] S436.421[农业科学—农业昆虫与害虫防治]