CAJ | 학술논문

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。
통과RT-PCR획득소맥황화협병독（ Wheat yellow mosaic virus， WYMV）양주분리물P2기인，병진행서렬분석。 P2기인편마구함유2635개핵감산，편마일개유875개안기산조성적분자량72 kDa적단백。재원핵표체체계중，해기인적전장표체교곤난，인차장P2기인적5′단화3′단각설계대약1 kb아극륭도원핵표체재체 pMAL-C2X중구건중조표체재체 MBP-P2N，MBP-P2C，경 IPTG유도재대장간균BL21（DE3）중표체MBP-P2N화MBP-P2C융합단백。 MBP-P2C융합단백경대량유도、순화、제비상응항혈청。용제비적항혈청가이유효검측WYMV병협중적 P2단백。면역효체금표기실험표명재감병WYMV적소맥협편세포막상내함체결구중발현유대량효체금과립특이성분포，표명P2단백가능여막상내함체결구유관。
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 .