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

豆科植物凝集素是最大的凝集素家族之一，它们在理化性质上彼此相似，尽管它们在碳水化合物特异性上不同。这些凝集素的碳水化合物结合位点由β折叠片上的2个保守氨基酸和2个环组成。其中一个环含有过渡金属、钙和锰，并将糖结合位点的氨基酸残基保持在所需位置。该环的氨基酸序列在这些凝集素的碳水化合物结合特异性中起重要作用。在编码羊蹄甲凝集素（BPA）的cDNA序列中，对应于该糖结合环的一部分进行了随机突变。用淘选法筛选在λ foo酵母表面表达的突变凝集素文库。分别分离出对甘露糖、N-乙酰半乳糖胺或N-乙酰葡糖胺具有亲和力的几个克隆。对怀槐凝集素（Maackia amurensis lectin，MAH）的cDNA进行随机突变，将突变后的cDNA导入pGEX-2 T质粒中，随机选择16个重组突变凝集素克隆进行特异性分析。结果表明，这些重组蛋白在糖结合结构域中含有不同的残基，并与抗MAH多克隆抗体发生强烈反应。用纯化的突变凝集素对人、牛、猪、马和鸡的红细胞进行血凝试验，结果表明，与野生型MAH相比，16个细胞的糖结合特异性发生了变化，但唾液酸酶处理细胞后，血凝活性没有显著降低。

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.