Redmond T M, Si J S, Barrett D J, Borst D E, Rainier S, Kotake S, Gery I, Nickerson J M
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, MD 20892.
Gene. 1989 Aug 1;80(1):109-18. doi: 10.1016/0378-1119(89)90255-2.
We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.
我们扩展了牛视网膜色素上皮细胞间类视黄醇结合蛋白（IRBP）的cDNA序列，并将其中一个测序的cDNA片段亚克隆到一个表达载体中。已确定了四个牛IRBP cDNA克隆的核苷酸（nt）序列。这些序列组装后覆盖了IRBP mRNA 3'近端的3629个nt，并编码IRBP的C末端551个氨基酸（aa）。该cDNA序列验证了从基因预测的内含子：外显子边界。将测序克隆之一lambda IRBP2的一个2kb EcoRI插入片段（编码IRBP的C末端136个aa）亚克隆到表达载体pWR590-1中。携带该质粒构建体pXS590-IRBP的大肠杆菌产生了一种融合蛋白，该融合蛋白包含β-半乳糖苷酶的583个N末端aa、三个接头aa残基、IRBP的136个C末端aa，并且由于终止抑制可能还有一些额外的C末端残基。这种86 kDa的融合蛋白通过去污剂/离液剂提取，然后通过反相高效液相色谱法纯化，在蛋白质免疫印迹上与抗牛IRBP发生交叉反应。这种蛋白在Lewis大鼠中诱导了实验性自身免疫性葡萄膜视网膜炎和实验性自身免疫性松果体炎，与真实的牛IRBP诱导的情况无法区分。因此，很明显，这种IRBP区域的生物活性，如免疫致病性所表现的，被融合蛋白保留了下来。
