IMMUNE-LIKE PROTEINS OF INVERTEBRATES

长寿的无脊椎动物在某种意义上是免疫无能的， 它们缺乏免疫系统的经典T和B细胞臂， 它们成功地应对微生物的挑战， 涉及致敏吞噬细胞的炎症机制。 这 该提案是根据先前关于“动物”的拨款（GM 30672）提出的 凝集素作为识别分子”，发现某些 被囊动物种的碳水化合物结合凝集素可能是 被鉴定为两个分子家族的成员， 关键免疫防御;即，急性期蛋白的 正五聚蛋白型（例如C反应蛋白）和 免疫球蛋白超家族 我们专注于类免疫识别 被囊动物的分子，因为这些原索动物物种是 是真正的脊椎动物的祖先，但缺乏复杂的 B和T细胞免疫的特征。 (1)我们设计了 用于纯化半乳糖结合蛋白的分级分离方案 被囊动物Didemnum candidum的凝集素，两种特异于 唾液缀合物和一个岩藻糖从被囊Halocynthia 梨形，一种来自七鳃鳗的唾液酸结合凝集素， 来自被囊Boltenia ovipera的分子， 与较低物种的Ig和抗 合成免疫球蛋白连接区肽。 纯化的 分子的物理化学特征将通过测定 a）天然和亚基分子量，B）碳水化合物和氨基 c）结合位点的数目和亲和力，以及 d）使用旋光法研究二级结构 色散和圆二色性测量。 (2)我们将 确定这些分子的氨基酸序列， 使用确定特异性的蛋白水解酶的肽， 化学裂解试剂，如CNBr，其分辨率为 用高效凝胶过滤色谱法 液相色谱法和反相肽色谱法。 将使用ABI脉冲液相对肽进行测序 测序仪 氨基酸序列将有助于详细描述 与已知的识别分子进行比较， 合成寡核苷酸探针以用于 分离相关基因。 (3)为了确保我们获得完整的 被囊动物和七鳃鳗凝集素的序列信息，我们将 运用分子生物学技术。 两个战略将 第一个是生产基因组DNA文库， 用合成的寡核苷酸探针筛选这些， 凝集素分子的可用序列。 第二策略 将产生cDNA文库， 表达载体然后将使用 纯化凝集素的抗体和合成探针。 DNA 将使用桑格双脱氧链测定法进行测序 法 我们将根据基因的数量来描述它们的特征 以及段的类型和这些元件的布置。

Long lived invertebrates are immunoincompetent in the sense that they lack the classical T and B cell arms of the immune system but they respond successfully to challenge with micro-organisms by inflammatory mechanisms involving sensitized phagocytes. This proposal follows from a previous grant (GM 30672) on "animal lectins as recognition molecules", finding that certain carbohydrate-binding lectins of tunicate species could be identified as members of two families of molecules known to be crucial to immune defense; namely, acute phase proteins of the pentraxin type (e.g. C-reactive proteins) and members of the immunoglobulin superfamily. We focus upon immune-like recognition molecules of tunicates because these protochordate species are ancestral to true vertebrates but are lacking in the sophisticated features of B and T cell immunity. (1) We have devised fractionation schemes for the purification of galactose-binding lectins of the tunicate Didemnum candidum, two lectins specific for sialoconjugates and one for fucose from the tunicate Halocynthia pyriformis, a sialic acid binding lectin from the lamprey and a molecule from the tunicate Boltenia ovipera that is serologically cross-reactive with Igs of lower species and with antibodies to synthetic immunoglobulin joining region peptide. The purified molecules will be characterized physicochemically by determining a) native and subunit molecular weights, b) carbohydrate and amino acid compositions, c) the number and affinity of binding sites, and d) investigations of secondary structure using optical rotatory dispersion and circular dichroism measurements. (2) We will determine the amino acid sequence of these molecules, preparing peptides using proteolytic enzymes of defined specificity and chemical cleavage reagents such as CNBr with the resolution of peptides by gel filtration chromatography using high performance liquid chromatography and by reverse phase peptide chromatography. The peptides will be sequenced using the ABI pulsed liquid phase sequencer. The amino acid sequence will facilitate detailed comparisons with known recognition molecules and enable the synthesis of synthetic oligonucleotide probes to be used in isolation of relevant genes. (3) To ensure that we obtain complete sequence information for the tunicate and lamprey lectins, we will employ the technology of molecular biology. Two strategies will be followed: the first is to produce libraries of genomic DNA and screen these with synthetic oligonucleotide probes predicted from the available sequence of the lectin molecules. A second strategy will be to produce cDNA libraries constructed using total mRNA in expression vectors will then be screened serologically using antibodies to the purified lectins and the synthetic probes. DNA will be sequenced using Sanger's dideoxy chain determination method. We will characterize the genes in terms of their number and types of segments and the arrangements of these elements.