暗纹东方纯CD8α基因克隆、原核表达与多克隆抗体制备

CD8是与Ⅰ型主要组织相容性复合体（MHCI）结合，是T细胞表面受体（TCR）的共受体．也是T淋巴细胞表面的重要标志物。该研究报道了暗纹东方纯胸腺等组织中克隆获得CD8αcDNA序列及其相应的基因组序列。克隆到的cDNA序列全长1061bp，包含1个657bp的开放读码框（ORF），编码218个氨基酸；其对应的基因组序列为1533bp，包含5个内含子和6个外显子。生物信息学分析表明，D8d蛋白质序列由信号肽、胞外可变区、铰链区、跨膜区和胞内区5部分组成。胞外区的可变区和铰链区部分各有两个高度保守的半胱氨酸残基，可能参与链内和链间二硫键的形成。氨基酸序列多重比对表明，暗纹东方鲍与其他鱼类的CD80α，特别是与鲽形目鱼类具有较高的同源性。为进一步研究暗纹东方纯CD8的生物学功能，构建了表达CD8α成熟肽胞外区的重组质粒，诱导表达出重组蛋白。以纯化的重组蛋白为抗原免疫大白兔，制备了抗血清。经间接ELISA法检测抗体效价表明。获得了高效价的特异性暗纹东方纯CD8c~抗体，为进一步研究CD8在鱼类淋巴细胞进化和适应性免疫中的作用奠定了基础。

CD8α gene in Takifugu fasciatus： cloning, expression and preparation of polyclonal antibody

In the fish species with T cellular immune function,CD8 molecules are important immune acceptor molecules.The cDNA sequence and its corresponding genomic sequence were cloned from the thymus of Takifugu fasciatus.The cDNA of Takifugu fasciatus CD8α was found to be 1 061 nucleotides long,the open reading frame(ORF) 657 bp long,and its corresponding genomic sequence 1 533 bp.The CD8α gene consisted of six exons separated by five introns and encoded by 218 amino acid residues with an estimated molecular weight of 22 Kda.The predicted primary structure of CD8α contained a signal peptide,an IgV-domain,a hinge region,a transmembrane region and the cytoplasmic tail.The two cysteine residues were involved in the V-domain of T.fasciatus CD8α capable of forming an intra-chain disulphide bond,and the other two cysteines in the hinge region are conserved in most of fishes responsible for CD8α’s ability of forming both homodimers and heterodimers.The protein structure of the T.fasciatus CD8α gene was similar to other fishes,especially to those of Pleuronectiformes.In order to further investigate the biological function of CD8,the CD8α fragment constructing the extracellular region of mature peptide was sub-cloned into pET28a(+) expression vector after digestion with a combination of Hind III and Xho I.The protein was expressed in bacteria by inducing and was purified.The anti-CD8α polyclonal antibody was prepared by immunization of rabbit with the purified fusion protein.The specific recognition anti-CD8α polyclonal antibody was further verified by ELISA analysis of the serum from pre-immunized and post-immunized rabbit.The preparation of CD8α antibody will greatly facilitate the identification and understanding of CD8 in fish lymphocyte populations and adaptive immunity.