猪胆囊收缩素-33基因同向四串联体的构建与表达

Cloning and prokaryotic exprssion of cholecystokinin-33 (CCK33) concatemer of pig

根据GenBank上公布的猪胆囊收缩素(choleystokinin,CCK)-33基因的cDNA序列设计特异引物,以猪十二指肠cDNA为模板,PCR扩增得到CCK33基因,并克隆入pMD18-T载体,利用引物中设计的一对同尾酶构建CCK33四串联体,并在大肠杆菌中成功表达.表达纯化蛋白制备抗原,得到多克隆抗体.Western blot显示重组蛋白具有良好的免疫原性.

Designed a pair of primers according to the published nucleotide sequence of pig cholecystokinin-33 （CCK33） gene from GenBank. Use the eDNA of superior duodenal flexure of pig as template to amplify CCK33 gene and cloned into pMD18-T vector. Its concatemer was constructed based on the fact that isocaudarner enzyme existed in primers. The recombinant CCK33 protein was expressed in E. coli BL 21 and purified successfully. The purified protein was used as an antigen to prepare polyclonal antibody. The result of western blot indicated that CCK33 concatemer has good immunogenicity.