【作者】 李永丽；

【导师】 王振跃； 吴茂森；

【作者基本信息】 河南农业大学 ， 植物病理学， 2004， 硕士

【摘要】 大麦黄矮病毒（Barley yellow dwarf virus, BYDV）是一种十分重要的禾谷类作物病毒。尽管有研究推测 BYDV 的 ORF4 与病毒在寄主体内的运动有关，但关于病毒在寄主体内的运转机制尚不了解。本研究首次对我国特有的BYDV-GPV株系的ORF4基因进行原核表达，并制备特异性的抗血清，成功用于 BYDV-GPV 侵染后燕麦体内表达的 17kDa 蛋白的检测，为进一步的研究奠定基础。 根据已测定的 BYDV-GPV 的相关序列设计合成含有适宜酶切位点的相应引物，通过 RT-PCR 方法扩增出ORF4 基因片段并将其插入到 pGEM-T Easy 质粒中，获得了重组质粒pGEM-T-O4。对基因片段进行序列分析结果表明，ORF4 全长 456 个核苷酸，可编码 152 个氨基酸，分子量为 17kDa 的蛋白质。pGEM-T-O4 经 Nde I和 BamH I 双酶切后插入到质粒 pET-5a 中获得原核表达载体pET-O4。经 IPTG 诱导基因表达及 SDS-PAGE结果分析，与作为对照的(i)未经诱导的含有pET-O4 质粒的和(ii)诱导的含 pET-5a 质粒的 BL21(DE3)pLysS 相比，诱导后的 pET-O4 中的 ORF4 基因在大肠杆菌 BL21(DE3)pLysS 中得到了特异性的表达，诱导表达的蛋白质分子量大小约为 17kDa，与通过基因序列推导的大小一致。试验对不同浓度的 IPTG、以及不同时间诱导的蛋白表达水平进行了研究，结果显示，不同浓度的 IPTG 对蛋白的诱导表达影响不大，而不同诱导时间对蛋白的诱导表达量有一定的影响，8h 以前时间越长表达量越高，但 8h 以后表达量的增长不明显。 将 ORF4基因编码的蛋白从聚丙烯酰胺凝胶上切下后，经圆盘电泳进行回收纯化。通过免疫家兔获得了 BYDV-GPV ORF4 基因非融合蛋白的特异性抗血清，用微量滴定法测定抗血清的效价为 1/2048。<WP=7>BYDV-GPV ORF4基因在 E.coli 中表达蛋白的 Western blot 检测结果表明，所制备的抗血清能与诱导的 pET-O4／BL21(DE3)pLysS 及作为阳性对照的 17kDa 蛋白发生特异性反应，而与阴性对照 pET-5a／BL21(DE3)pLysS 无特异性反应。BYDV-GPV 侵染燕麦后的 Westernblot 分析结果表明，所获得的抗血清与接毒１d、２d、３d 和４d 的燕麦叶片中提取的蛋白样品均呈阳性反应，而与未接毒的燕麦叶片中提取的蛋白样品不产生任何阳性反应，并且随着接毒天数的增加反应呈增强趋势。更多还原

【Abstract】 Barley yellow dwarf virus (BYDV) is one of the mosteconomically important cereal viruses. Up to now, the mechanism of BYDV movingin vivo isn’t clear, although ORF4 gene of BYDV was proposed being related to virusmovement in hosts. In this study, ORF4 gene of BYDV-GPV was cloned andexpressed in Escherichia coli. The antiserum against protein expressed by BYDV-GPV ORF4 was raised and used in detection of the 17kDa protein of BYDV-GPV ininfected oats. Fragment of ORF4 was amplified through RT-PCR after the primers beendesigned and synthesized according to the published nucleotide sequence. Thefragment was inserted into pGEM-T Easy vector and the recombinant plasmid wasobtained and named of pGEM-T-O4. Sequence analysis showed that ORF4 contains456 nucleotides that can code a protein of 152 amino acids with Mr about 17kDa.pET-O4, the prokaryotic expression plasmid (containing ORF4), was obtained byinserting the fragment, when it was recovered from pGEM-T-O4 by digested withNde I and Bam HI, into pET-5a plasmid. After induced with IPTG, the proteins wereanalyzed via SDS-PAGE. 17kDa protein was expressed in BL21(DE3)pLysS whichcontaining pET-O4 plasmid, compared with that of not induced pET-O4 and inducedpET-5a. The expression results of pET-O4 induced with different concentration ofIPTG or in different time course showed that the concentration of IPTG had noobvious effect on the induced expression, but there was a stronger expression alongwith the time increase before 8 hours. The antiserum with titer of 1:2048 was obtained from rabbit after injections with 68<WP=74>purified 17kDa protein, which had a specific reaction with 17kDa protein, purified orinduced in pET-O4/BL21(DE3)plysS. The antiserum was also used to detect theexistence of 17kDa protein in oat after infected with BYDV-GPV. The result showedthat the antiserum had a positive reaction with all of the samples of infected for 1 day,2 days 3 days and 4 days, and the reaction was stronger when time prolonged afterinfection.更多还原