侵染浙贝母的百合斑驳病毒CP基因的原核表达及抗血清制备  

Prokaryotic Expression of LMoV CP Gene and Preparation of Its Antiserum

作　　者：韦传宝[1,2] 姚厚军[1] 杨宇[1] 刘春云[1] 

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

出　　处：《中药材》2011年第8期1182-1186,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(KJ2010A328KJ2011A272)

摘　　要：目的:制备抗百合斑驳病毒(Lily mottle virus,LMoV)CP血清,为研究侵染不同植物LMoV之间的血清学关系以及大田检测LMoV奠定基础。方法:根据Genbank报道的百合斑驳病毒CP基因序列设计引物,扩增其外壳蛋白基因并分析序列。将LMoV 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,采用ELISA分析抗体能否与天然LMoV病毒结合。结果:LMoV的CP与目前已报道的LMoV不同分离物CP基因核苷酸序列同源性为95%～99%,氨基酸序列同源性为98%～100%。制备的抗体对CP具有高度特异性,且能够与天然LMoV病毒离子结合。结论:本研究制备的抗血清可以作为百合斑驳病毒的检测。Objective： To prepare antiserum against the CP of Lilg mottle virus（LMoV）.Methods： Specific primer was designed according to Genbank to amplify CP gene of LMoV of Fritillaria thumbergii and its sequence was analyzed.Then the CP gene was inserted into pSBET and expressed in Escherichia coli BL21（DE3）plys E strain.The objective 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 their specificity was determined by Western blot.The ability to combine with nature LMoV particles was confirmed by ELISA analysis.Results： LMoV CP gene shared 95%-99% nucleotide identities and 98%-100% amino acid identities with the CP genes reported on Genbank.The antiserum was special to LMoV CP and IgG against LMoV could combine LMoV particles.Conclusion： The antiserum prepared in this study is suitable for LMoV detection.

关 键 词：浙贝母 百合斑驳病毒 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]