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

豆类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，但它们的碳水化合物特异性不同。这些凝集素的碳水化合物结合位点由2个保守的氨基酸和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异性中起重要作用。在紫荆凝集素（BPA）的cDNA编码部分引入了与该碳水化合物结合环对应的随机突变。用筛选法筛选在噬菌体表面表达的突变凝集素文库。对甘露糖、n -乙酰半乳糖胺和n -乙酰氨基葡萄糖胺分别具有亲和力的克隆进行了分离。将随机突变的dna导入到Maackia amurensis凝集素（MAH）编码的cDNA中，并将突变的cDNA导入pGEX-2T质粒中。随机选取16个重组突变凝集素克隆进行特异性研究。这些化合物在糖结合域含有不同的残基，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细胞中表达，与野生型MAH相比，对人、牛、猪、马和鸡红细胞进行了血凝试验。虽然与MAH相比，唾液酸酶处理的细胞并没有引起血凝活性的显著降低，但在16个结肠中碳水化合物结合特异性的变化被揭示出来。

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.