三黄鸡CD8α/βcDNA克隆与表达及其多克隆抗体的制备  

Cloning,expression and production of polyclonal antibodies in extracellular regions of chicken CD8α/β

作　　者：唐秀山[1] 王磊[1] 廖定为[1] 蒋一男[1] 夏春[1] 

机构地区：[1]中国农业大学动物医学院,北京100094

出　　处：《中国农业大学学报》2007年第5期5-9,共5页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(30671566)

摘　　要：为了获得鸡CD8α/β蛋白及其多克隆抗体,进一步研究其结构与功能,本实验从三黄鸡cDNA文库中克隆了其CD8α/β胞外区片段,构建了pQE30/ChCD8α/β原核表达系统,并经诱导表达、亲和层析纯化,获得了纯化的重组蛋白(rChCD8α/β),然后用rChCD8α/β分别免疫Balb/c小鼠制备了其多克隆抗体。结果表明:本实验克隆的ChCD8α长483 bp、编码161个氨基酸、可在E.coliJM109中高效表达,蛋白质分子质量为24.0 ku,表达量占菌体蛋白量的25.0%,由该蛋白质所制备的多克隆抗体抗rChCD8α的ELISA效价为1∶1 024 000;另外,克隆的ChCD8β长447 bp,编码149个氨基酸,蛋白质的分子质量为18.5 ku,表达量占菌体蛋白量的20.0%,抗rChCD8β多克隆抗体ELISA效价为1∶64 000。本实验所得鸡CD8α/β的多克隆抗体将用于四聚体研究。The aim of this research was to prepare proteins and polyclonal antibodies for further study of the structure and function of chicken CD8α /β. The extracellular regions of CD8α /β chains were amplified by PCR and expression systems （pQE30/ChCD8α /β） were constructed and induced. The recombinant protein （rChCD8α /β） was purified to immunize the Balb/c mice. The resulting cloned ChCD8α had 483 bp in length, encoding 161 amino acid residues. SDS-PAGE analysis showed that the molecular weight was 24.0 ku, and the mounts of the recombinant protein was 2.5.0% of total mass of bacterial protein. ELISA analysis showed that the titer of polycIonal anti-ChCD8α was about 1 ： 1 024 000. The cloned ChCD8α had 447 bp in length, encoding 149 amino acid residues. The SDS-PAGE analysis showed that the molecular weight was 18.5 ku, and the mounts of the recombinant protein was 20.0% of total mass of bacterial protein. ELISA analysis showed that the titer of polycIonal anti-ChCD8α was about 1：64 000. Antibodies provided from the research can be applied to the further study of tetramer.

关 键 词：鸡 CD8Α CD8β 原核表达 多克隆抗体 

分 类 号：Q785[生物学—分子生物学] S831[农业科学—畜牧学]