CARBOHYDRATE BINDING PROTEIN OF B. JAPONICUM

本项目拟研究BJ 38的结构和活性， 从慢生根瘤菌的提取物中纯化的凝集素。 这种蛋白质 与含有乳糖（Lac）或半乳糖（Gal）的糖缀合物结合。 C-2位Gal衍生物、半乳糖胺和N-乙酰基-D-半乳糖胺结合 亲和力低得多。 这种特异性与 完整根瘤菌在四种碳水化合物特异性结合测定中的表达：（a） （B）与培养的细胞的异型结合 大豆（SB-1）细胞;（c）对大豆根的异型粘附;和（d） 同型自身凝集。 这些观察结果表明 所有这些结合活性均由相同的组分介导， 而BJ 38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]为了证明和定量纯化的BJ 38与大豆的结合， 细胞和B。japonicum细胞; [b]为了测试与大豆细胞结合的可溶性BJ 38将 阻止B的粘附。与细菌结合BJ 38将 阻断自身凝集; [c]为了测试是否有针对 BJ 38将抑制与大豆细胞的异型结合和同型结合。 自体凝集; [d]分析BJ 38在B细胞中的表达和定位。日本 细胞; [e]进行BJ 38的分子克隆; [f]确定BJ 38蛋白质的部分氨基酸序列， 水平，并从DNA序列中推导出完整的氨基酸序列; [g]为了测试是否可以在突变体中恢复碳水化合物特异性结合， B。通过与BJ 38基因的互补作用，获得了一株日本血吸虫的真核表达载体。

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.