鸭MHCⅡα基因的表达纯化与抗体制备  

Expression,purification and antibody preparation of duck MHCⅡα gene

作　　者：张舒婕[1] 邓干臻[1] 龚炎长[1] 杨庆磊[1] 胡福利[1] 杨桓[1] 彭秀丽[1] 

机构地区：[1]华中农业大学教育部动物遗传育种与繁殖重点实验室,湖北武汉430070

出　　处：《中国兽医学报》2009年第4期464-467,共4页Chinese Journal of Veterinary Science

基　　金：湖北省"十一五"重大科攻关资助项目(2006AA202-A03)

摘　　要：根据NCBI上已发表的序列自行设计引物,通过RT-PCR从北京鸭脾脏的总RNA中扩增得到鸭MHCⅡα基因,将其克隆至pMD18-T,经酶切分析及序列测定鉴定后,进一步亚克隆至原核表达载体pET-28a。转化大肠杆菌BL21中诱导表达。电洗脱纯化蛋白,用于免疫昆明小鼠制备多克隆抗体。结果表明:克隆了鸭MHCⅡα基因,大小为633 bp,经核苷酸测序与已登录的基因序列同源性为99%;成功构建了原核表达载体,融合蛋白得到了高效表达且纯化后纯度可达90%,制备的鼠抗鸭MHCⅡα多克隆抗体经酶联免疫吸附试验(ELISA)与免疫印迹法(Western-blot)证实了抗体的效价高、特异性强,研究结果为研究鸭MHCⅡ分子奠定了基础。Aecroding to mRNA gene sequence registered in GenBank,a pairs of sepcifie primers for the genes of chain α of MHC Ⅱ of duck were designed and synthesized. Using total RNA from duck spleen, the target gene fragment was obtained by RT-PCR, then cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert was subcloned into the expression vector pET-28a. Transformed into E. cull BL21 ,the recombination plasmid were induced to obtain the interest protein. Use electroeluting purified the protein to immunize the mouse for preparing polyclonal antibody. The results showed we cloned the MHC Ⅱα gene, the obtained 633 bp fragment has 99% identities to the previously identified duck MHC Ⅱ alpa at nucleotide level; successfully constructed the porkaryotic expression vector and the protein expressed efficiently. The protein purified and purity reached 90%. A high titer and specific antibody has been prepared by purified protein,it was proved by the methods of ELISA and Western blot. All this makes it possible to do further studies of duck MHC Ⅱ gene.

关 键 词：:鸭 MHCⅡα基因 克隆 原核表达 纯化 多克隆抗体 

分 类 号：S852.2[农业科学—基础兽医学]