用标记磁微粒技术克隆牛CD14基因

为获得牛ＣＤ１４ 的全长编码基因，以探讨牛ＣＤ１４ 基因结构与功能的关系，进一步阐明其作用机理，我们用抗牛ＣＤ１４ 的单克隆抗体（ｍＡｂ）ＣＣＧ３３ 标记的磁微粒作为探针，从转染有牛肺泡巨噬细胞ｃＤＮＡ文库的ＣＯＳ７细胞中进行筛选。并对获得的牛ＣＤ１４ ｃＤＮＡ进行了克隆和序列分析。结果表明，牛ＣＤ１４ 基因全长为１１２２ ｂｐ，共编码３７３ 个氨基酸，其中含有信号肽序列和３ 个可识别的Ｎ连接糖基化位点，但缺少穿膜结构域及细胞内区。证实牛ＣＤ１４ 以糖基磷脂酰肌醇（ＧＰＩ） 锚定在靶细胞膜上；其氨基酸序列与人和小鼠ＣＤ１４ 的同源性分别为７３－５ ％ 和６１－４ ％ ，三者共有的保守序列约为５５％ 。

Molecular cloning of bovine CD14 gene by labeled magnetic bead technique

The bovine CD14 cDNA was obtained from the COS 7 cells which were transfected with the cDNA library of bovine alveolar macrophages by using the magnetic beads coated with monoclonal antibody against bovine CD14 as a probe The full length of coding bovine CD14 gene is 1122 bp coding for 373 amino acids It contains the signal peptide sequence and three potential N linked glycosylation sites, but lacks transmembrane domain and intracellular region It is suggested that bovine CD14 is anchored on the cell membrane through glycosylphosphatidylinositol(GPI) The identities of its amino acid sequence to that of human and murine CD14 are 73 5%and 61 4%respectively, and the common conserved sequences among the three species are about 55%