Luo D, Müller H, Tang X B, Hobom G
Department of Medicine, University of Alberta, Edmonton, Canada.
J Gen Virol. 1994 Jun;75 ( Pt 6):1267-80. doi: 10.1099/0022-1317-75-6-1267.
Budgerigar fledgling disease virus (BFDV) represents the first non-mammalian member of the polyomavirus genus and possesses uncommon structural and biological properties. Recombinant baculoviruses were constructed to express BFDV small t antigen, large T antigens, as well as a large T deletion mutant Td and beta-galactosidase-Td fusion proteins to high levels in infected insect cells. A recombinant virus containing a genomic copy of the BFDV early region was used for small t antigen expression, and corresponding intron-deleted cDNAs for production of large T antigen derivatives. Recombinant T as well as authentic T antigen proteins from infected chicken embryo fibroblasts were purified using both immunoaffinity and DNA affinity column chromatography. We present evidence that the large T antigen interacts specifically with DNA sequences present in the non-coding region of BFDV; by indirect DNA immunoprecipitation mapping and DNase I footprinting, four regions including 12 DNA-binding sites have been determined that cover most of the BFDV non-coding region. The T antigen binding pattern observed suggests a protein-DNA interaction system considerably different from those of simian virus 40 and other polyomaviruses.
虎皮鹦鹉幼雏病病毒（BFDV）是多瘤病毒属的首个非哺乳动物成员，具有独特的结构和生物学特性。构建了重组杆状病毒，以便在受感染的昆虫细胞中高水平表达BFDV小t抗原、大T抗原，以及大T缺失突变体Td和β-半乳糖苷酶-Td融合蛋白。含有BFDV早期区域基因组拷贝的重组病毒用于小t抗原表达，使用相应的缺失内含子的cDNA来生产大T抗原衍生物。使用免疫亲和柱层析和DNA亲和柱层析，对来自受感染鸡胚成纤维细胞的重组T以及天然T抗原蛋白进行了纯化。我们提供的证据表明，大T抗原与BFDV非编码区中存在的DNA序列特异性相互作用；通过间接DNA免疫沉淀图谱分析和DNase I足迹分析，已确定了包括12个DNA结合位点的4个区域，这些区域覆盖了大部分BFDV非编码区。观察到的T抗原结合模式表明，其蛋白质-DNA相互作用系统与猿猴病毒40和其他多瘤病毒的系统有很大不同。
