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Construction of novel type lectins having unique sugar-binding specificities by using lambda foo phage display system, and their use for gene targetting

利用 lambda foo 噬菌体展示系统构建具有独特糖结合特异性的新型凝集素及其在基因打靶中的应用

基本信息

批准号：
08672502
负责人：
YAMAMOTO Kazuo
金额：
$ 1.47万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672502/
关键词：
lectin phage display system lambda foo シアル酸ファージディスプレイファージ

项目摘要

Legume lectins are one of the largest lectin families and they resemble each other in their physico-chemical properties although they differ in their carbohydrate specificities. The carbohydrate-binding sites of these lectins consist of 2 conserved amino acids on beta pleated sheets and 2 loops. One of these loops contains transition metals, calcium and manganese, and keep the amino acid residues of the sugar-binding site at the required positions. Amino acid sequences of this loop play an important role in the carbohydrate-binding specificities of these lectins. Random mutation was introduced in a part of cDNA coding Bauhinia purpurea lectin (BPA) which correspond to this carbohydrate-biniding loop. Mutated lectin library expressed on the surface of lambda foo phages were screened by the panning method. Several clones having the affinity for mannose, N-acetylgalactosamine, or N-acetylglucosamine were isolated, respectively. Random mutation was also introduced into the cDNA encoding Maackia amurensis lectin (MAH) and the mutated cDNAs were introduced into plasmid of pGEX-2T.Sixteen clones of recombinant mutated lectins were rondomlly chosen to investigate their specificities. All of these were found to contain different residues in sugar-binding domain, and reacted strongly with anti-MAH polyclonal antibody. Hemagglutination of the purified mutated lectins expressed in E.coli cells were tested against human, bovine, pocine, equine and chicken erythrocytes, as compared to wild type MAH.Variations in carbohydrate-binding specificities aong sixteen colnes were revealed though sialidase treatment of cells did not cause a significant decrease in hemagglutinating acitivity conparde to MAH.

豆科凝集素是最大的凝集素家族之一，它们的物理化学性质相似，尽管它们的碳水化合物特性不同。这些凝集素的碳水化合物结合部位由2个保守的氨基酸组成，包括2个β折叠片层和2个环。其中一个环含有过渡金属、钙和锰，并将糖结合部位的氨基酸残基保持在所需的位置。该环的氨基酸序列在这些凝集素的碳水化合物结合特性中起着重要作用。在编码紫荆花凝集素(BPA)的部分cDNA中引入了随机突变，该突变对应于这个碳水化合物结合环。用淘选法筛选表达于Lambda Foo噬菌体表面的突变凝集素文库。分离到与甘露糖、N-乙酰氨基半乳糖和N-乙酰氨基葡萄糖有亲和力的克隆。将随机突变的重组山玛瑙凝集素(MAH)基因导入pGEX-2T载体，随机筛选出16个重组突变的凝集素克隆，研究其特异性。所有这些化合物都含有不同的糖结合域残基，并与抗MAH多克隆抗体发生强烈反应。纯化的突变凝集素在大肠杆菌中表达，与野生型MAH相比，与人、牛、麻鸡、马和鸡的红细胞的血凝活性进行了比较，发现16种细胞的糖类结合特性存在差异，尽管唾液酸酶处理细胞并没有引起与MAH相比的血凝活性的显著下降。