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2635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 P 2 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 .
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