Purification, Structure Determination, and Gene Cloning For Bioactive Biopolymers of Marine Invertebrates

海洋无脊椎动物生物活性生物聚合物的纯化、结构测定和基因克隆

基本信息

批准号：
01470131
负责人：
KAMIYA Hisao
金额：
$ 3.14万
依托单位：
School of Fisheries Sciences, Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

(1) Purification and structure determination of bioactive biopolymers : The amino acid sequence of a galactose-specific lectin from acorn barnacle, Megabalanus rosa, was been determined. The lectin (BRA-2, mol. wt. 140,000) sequence was determined by peptides derived from the prote in digestion and and fragments produced by cyanogen bromide cleavage. The amino acid sequence of the lectin was compared with the sequence of one (BRA-3, mol. wt. 64,000) of M. rosa. They are distinct molecules in a significant homology in their amino acid sequences. The two intrachain disulfids bonds are well conserved through all invertebrate lectins and calcium-dependent animal lectins.A novel galactose-sepcific lectin showing a molecular weight of 78,000 was first isolated from a soft coral, Sinularia sp. The lectin induced the binding between macrophage and tumor cells.The antimicrobial protein (Julianin S) was detected in the fetid secretion of a sea hare, Aplysia juliana. It inhibited the growth of both Gram-positive and Gram-negative bacteria. It also inhibited in vitro growth of L1210 cells by 50% at a concentration of 8 ng protein/ml.(2) cDNA cloning and DNA sequencing : Total cellular RNA from the acorn barnacle was isolated from the specimen collected in summer when BRA-3 in the barnacle showed the highest content. Poly (A) RNA from the was used for cDNA cloning. After screening by colony hybridization, three clones, BRA 3-1, 3-2, and 3-4, showing a size of 0.5-0.7 kilobase were selected for DNA sequence determination. These clones contained the coding region to the whole sequence of BRA-3. The coding region was preceded by a signal peptide region (24 amino acid residues). Replacement of Arg to Lys at position 122 in the BRA-3 molecule was also confirmed the DNA sequencing. Northern analysis revealed that the seasonal change in the BRA-3 content was preceded by the change of mRNA in the acorn barnacle.

（1）确定了生物活性生物聚合物的纯化和结构测定：确定了来自Megabalanus rosa橡子的半乳糖特异性凝集素的氨基酸序列。凝集素（Bra-2，mol。wt。140,000）序列是由源自消化中蛋白的肽和氰基溴化物裂解产生的片段。将凝集素的氨基酸序列与M. rosa的一（bra-3，摩尔64,000）的序列进行了比较。它们在其氨基酸序列中的显着同源性中是不同的分子。这两个内部二硫键通过所有无脊椎动物凝集素和钙依赖性动物凝集素得到很好的保守。一种新型的半乳糖 - 分离凝集素，显示分子量为78,000，首先是从软珊瑚sinularia sp中分离出来的。凝集素诱导的巨噬细胞和肿瘤细胞之间的结合。在海兔，阿波利西亚朱莉安娜的恶臭分泌中检测到抗菌蛋白（朱利安蛋白S）。它抑制了革兰氏阳性和革兰氏阴性细菌的生长。它还以8 ng蛋白/ml的浓度抑制50％的L1210细胞的体外生长。（2）（2）cDNA克隆和DNA测序：从夏季收集的标本中分离出来自橡子藤壶的总细胞RNA，而Bra-3在Barnacle中显示出最大的内容。来自该的poly（a）RNA用于cDNA克隆。通过菌落杂交筛选后，选择了三个克隆，胸罩3-1、3-2和3-4，显示了0.5-0.7千行酶的大小以确定DNA序列。这些克隆在BRA-3的整个序列中包含编码区域。编码区域之前是信号肽区（24个氨基酸残基）。还确认了在BRA-3分子中的位置122处替换为LYS的LYS。北方分析表明，BRA-3含量的季节性变化是在橡子棒上变化的mRNA。