小麦黄花叶病毒P2蛋白原核表达、抗血清制备及其在感病小麦细胞中的定位  

Subcellular immuno-localization of P2 protein encoded by Wheat yellow mosaic virus RNA2,using poly-clonal antibody raised against prokaryotic expressed P2 fusion protein

作　　者：杨狄[1,2] 周燕茹[2] 谢礼[2] 孙丽英[2] 陈剑平[2] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036 [2]浙江省农业科学院病毒学与生物技术研究所,浙江杭州310021

出　　处：《浙江农业学报》2014年第3期742-747,共6页Acta Agriculturae Zhejiangensis

基　　金：国家小麦产业技术研究体系专项(CARS-3-1);公益性行业专项(201303021)

摘　　要：通过RT-PCR获得小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)扬州分离物P2基因,并进行序列分析。P2基因编码区含有2 635个核苷酸,编码一个由875个氨基酸组成的分子量72 kDa的蛋白。在原核表达体系中,该基因的全长表达较困难,因此将P2基因的5'端和3'端各设计大约1 kb亚克隆到原核表达载体pMAL-C2X中构建重组表达载体MBP-P2N,MBP-P2C,经IPTG诱导在大肠杆菌BL21(DE3)中表达MBP-P2N和MBP-P2C融合蛋白。MBP-P2C融合蛋白经大量诱导、纯化、制备相应抗血清。用制备的抗血清可以有效检测WYMV病叶中的P2蛋白。免疫胶体金标记实验表明在感病WYMV的小麦叶片细胞膜状内含体结构中发现有大量胶体金颗粒特异性分布,表明P2蛋白可能与膜状内含体结构有关。P2 gene of Wheat yellow mosaic virus（ WYMV） was amplified by RT-PCR from WYMV infected wheat leaves. Sequence analysis indicated that P2 gene constituted of 2 635 nts,encoding a protein of 875 amino-acids（ AA） with estimated molecular weight of 72 kDa. Due to its large protein molecular weight,it is difficult to be expressed in prokaryotic expression system. Thus,5＇-terminal and 3＇-terminal parts of P2 gene（ 1 kb each） were subcloned into the prokaryotic expression vector pMAL-C2X,respectively. Subsequently,the expressed MBP-P2N and MBP-P2C fusion protein were induced by IPTG in E. coli BL21（ DE3）. The abundantly induced MBP-P2C infusion protein then was purified,and the rabbit antiserum against to this protein was prepared. Experiments indicated that this antiserum can be used to detect the P2 protein in WYMV infected wheat leaves by Western blotting and immuno-gold labeling. It was shown that WYMV P2 protein was located in subcellular membranous body,indicating that this protein might be associated with membrane-derived inclusions.

关 键 词：小麦黄花叶病毒 P2基因 原核表达 抗血清制备 免疫胶体金标记 

分 类 号：S435.12[农业科学—农业昆虫与害虫防治]