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，但 他们通过以下方式成功应对微生物的挑战 致敏吞噬细胞的炎症机制。这 这项建议是根据上一次拨款(通用汽车30672)提出的。 凝集素作为识别分子，发现这一点 被囊物种的碳水化合物结合凝集素可能是 被鉴定为已知的两个分子家族的成员 对免疫防御至关重要；即，急性时相蛋白 五肽类(例如C-反应蛋白)和 免疫球蛋白超家族。我们专注于免疫类识别 因为这些原索动物的分子是 是真正的脊椎动物的祖先，但缺乏成熟的 B细胞和T细胞免疫的特点。(1)我们设计了 纯化半乳糖结合蛋白的分级方案 被囊生毛竹的凝集素，两种专用于 唾液酸偶联物和一种来自被囊藻的岩藻糖 Yriformis，七鳃鳗唾液酸结合凝集素和 来自卵囊状牛肝菌的分子，是血清学的 与低等物种的免疫球蛋白及抗 合成免疫球蛋白连接区多肽。纯净的 分子的物理化学特征将通过确定 A)天然分子量和亚单位分子量，b)碳水化合物和氨基 酸组成，c)结合位点数和亲和力，以及 D)用旋光法研究二级结构 色散和圆二向色性测量。(2)我们会 测定这些分子的氨基酸序列，准备 使用特定特异性的蛋白水解酶和 化学切割试剂，如CNBr，其分辨率为 高效凝胶过滤层析分离多肽的研究 采用高效液相色谱和反相多肽层析法。 这些多肽将使用ABI脉冲液体相进行测序 定序器。氨基酸序列将有助于详细说明 与已知的识别分子进行比较，使 用于生物合成的寡核苷酸探针的合成 相关基因的分离。(3)确保我们获得完整的 关于被囊藻和七鳃鳗凝集素的序列信息，我们将 运用分子生物学技术。有两个战略将 遵循：第一个是建立基因组DNA文库和 用人工合成的寡核苷酸探针对这些基因进行筛选 凝集素分子的可用序列。第二种战略 将产生用总mRNA构建的cDNA文库 然后对表达载体进行血清学筛选 纯化的凝集素和合成的探针的抗体。脱氧核糖核酸 将使用桑格的双脱氧链测序法进行测序 方法。我们将根据基因的数量来描述它们的特征 以及分段的类型和这些元素的排列。

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.