CARBOHYDRATE BINDING PROTEIN OF B. JAPONICUM

本项目拟研究BJ38的结构和活性。 从慢生根瘤菌提取物中提纯的凝集素。这种蛋白质 与含有乳糖(Lac)或半乳糖(Gal)的糖偶联物结合。 半乳糖胺和N-乙酰-D-半乳糖胺结合的半乳糖C-2衍生物 亲和力要低得多。这种特异性与 四种碳水化合物特异性结合试验中的完整根瘤菌：(A) 乳胶-琼脂糖珠的吸附；(B)异型结合培养 大豆(SB-1)细胞；(C)与大豆根的异型黏附；和(D) 同型自体凝集。这些观察表明有可能 所有这些结合活性都是由相同的成分介导的(S) 而BJ38是碳水化合物特异性结合的关键候选者。 拟议研究的具体目标是： [A]证明和定量纯化的BJ38与大豆的结合 细胞和B.Japan onicum细胞； [B]测试可溶性BJ38与大豆细胞结合的可能性 阻断日本血吸虫黏附和BJ38与细菌结合将 阻断自身凝集反应； [c]检测单价抗体Fab片段是否针对 BJ38会抑制与大豆细胞的异型结合和同型 自身凝集； [D]分析BJ38在日本血吸虫中的表达和定位 细胞； [E]进行BJ38的分子克隆； [F]测定BJ38蛋白的部分氨基酸序列 并从DNA序列中推导出完整的氨基酸序列； [g]测试突变体中是否可以恢复碳水化合物特异性结合 通过与BJ38基因的互补，获得了BJ38基因。

This project proposes to study the structure and activities of BJ38, a lectin purified from extracts of Bradyrhizobium japonicum. This protein binds to glycoconjugates containing lactose (Lac) or galactose (Gal). Derivatives of Gal at C-2, galactosamine and N-acetyl-D-galactosamine bound with much lower affinity. This specificity correlated with the behavior of intact Rhizobium in four carbohydrate-specific binding assays: (a) adsorption to Lac-Sepharose beads; (b) heterotypic binding to cultured soybean (SB-1) cells; (c) heterotypic adhesion to soybean roots; and (d) homotypic autoagglutination. These observations indicate the possibility that all of these binding activities were mediated by the same component(s) and the BJ38 is a key candidate for the carbohydrate-specific binding. The specific objectives of the proposed research are: [a] to demonstrate and quantitate the binding of purified BJ38 to soybean cells and to B. japonicum cells; [b] to test the possibility that soluble BJ38 bound to soybean cells will block adhesion of B. japonicum and that BJ38 bound to the bacteria will block autoagglutination; [c] to test whether univalent Fab fragments of antibodies directed against BJ38 will inhibit heterotypic binding to soybean cells and homotypic autoagglutination; [d] to analyze the expression and localization of BJ38 in B. japonicum cells; [e] to carry out molecular cloning of BJ38; [f] to determine the partial amino acid sequence of BJ38 at the protein level and to derive the complete amino acid sequence from the DNA sequence; [g] to test whether carbohydrate-specific binding can be restored in mutant B. japonicum by complementation with the BJ38 gene.