风雨花甘露糖结合凝集素基因克隆与序列分析

单子叶甘露糖结合凝集素具有抑制人类免疫缺陷病毒的活性。风雨花 (Zephyranthes grandiflora)球茎中含有亚基分子量为 12 KD的甘露糖结合凝集素 (ZGA)。运用同源克隆的方法设计简并引物 ,通过 3'和 5 'RACE技术 ,从风雨花总 RNA中克隆了编码此凝集素的全长 c DNA序列 ,其开放阅读框长 4 92 bp,编码 16 4个氨基酸 ,包含信号肽序列、成熟蛋白序列和 C-末端剪切序列的前体蛋白。成熟蛋白由 10 6个氨基酸残基组成 ,分子量为 11.6KD。成熟蛋白在氨基酸水平上与石蒜凝集素、雪花莲凝集素、水仙凝集素和君子兰凝集素分别有 71.8%、81%、81.8%和 84 .5 %的同源性 ;在 Blocks数据库中检索 ZGA蛋白氨基酸序列的结构域 ,发现有 3个凝集素功能结构域 ,并具有 3个典型的甘露糖专一结合位点盒 (QDNY)

Molecular Cloning and Analysis of a Monocot Mannose-binding Agglutinin from Zephyranthes Grandiflora(Family Amaryllidaceae)

The monocot mannose-binding lectin can inhibit HIV from infecting the target cells. The total RNA of Zephyranthes grandiflora was extracted and reversely transcribed into cDNA. Degenerate primers were designed based on the conserved regions of other monocot mannose-binding agglutinins by homology alignment. The 694bp full-length cDNA of Zephyranthes grandiflora agglutinin (ZGA) was cloned by RT-PCR, 3'and 5'RACE (rapid amplification of cDNA ends). The start codon and stop codon of ZGA were at 37-39bp and 529-531bp respectively. The NCBI Blast analysis result showed that ZGA gene encoded a protein precursor with signal peptide, mature protein and C-terminal cleavage sequence. The mature ZGA protein contained 106 amino acids residues and its molecular weight was 11.6KD. The percentages of identity of the deduced mature ZGA protein with those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin and Clivia miniata agglutinin were 71.8%,81%,81.8%and 84.5%, respectively. Blocks analysis revealed that ZGA had three functional domains and three mannose-binding boxes (QDNY).