鹅IL-18基因的克隆和原核表达及其多克隆抗体的制备  

Cloning and prokaryotic expression of goose interleukin-18 gene and the preparation of polyclonal antibody to recombinant goose IL-18

作　　者：郑海华[1] 韩先干[1] 尹会方[1] 周蕾[1] 柯艳坤[1] 黄青云[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2010年第3期274-278,共5页Chinese Veterinary Science

基　　金：广东省自然科学基金项目(32277)

摘　　要：参考GenBank中鸡、火鸡和鸭IL-18基因序列,设计了1对特异引物,以提取的鹅脾细胞总RNA为模板,应用RT-PCR扩增、克隆鹅IL-18基因中间的333bp序列。序列分析结果表明,其与鸭IL-18基因对应序列相似性最高,达99%。参考鸭IL-18全基因序列设计上、下游引物各3条,以梯度PCR探索扩增、克隆鹅IL-18全基因。以上述策略成功扩增、克隆了3个广东地方杂交品种鹅的IL-18基因。序列分析表明,这3个杂交品种鹅的IL-18基因序列一致,长642bp,含一个603bp的ORF,编码200个氨基酸,分子质量为23.2ku。ORFN端的30个氨基酸为前导序列,其余170个氨基酸为功能蛋白序列。在GenBank中注册了第一条鹅的IL-18基因序列,登录号为EF159728。随后,构建了鹅IL-18基因ORF的重组原核表达质粒pET-GIL-18,经诱导表达、纯化获得重组鹅IL-18融合蛋白,并以其制备了兔抗重组鹅IL-18多克隆抗体。According to duck,turkey and chicken IL-18 gene sequences available in GenBank,a pair of primers was designed and synthesized to clone the middle sequence of goose IL-18（GIL-18） gene about 333bp by RT-PCR from the total RNA of goose spleen lymphocyte.The sequence analysis showed that the nucleotide homology of the middle sequence between goose and duck was the highest 99%.Then according to the duck IL-18 gene sequence,three upstream and downstream primers were designed and a battery testing of RT-PCR was carried out to clone goose full-length IL-18 gene.The results indicated that the goose IL-18 gene sequence about 642bp from three breeds geese in Guangdong were amplified,cloned and sequenced successfully.And the three sequences were the same one,including a ORF about 603bp.The ORF that was 23.2ku in molecular weight encoded 200 amino acids.The leader sequence was 30 amino acids at the N-terminus,and the other 170 amino acids was functional sequence.This sequence was registered in GenBank（EF159728） for the first time.The recombinant expression plasmid pET-GIL-18 was constructed.Then the recombinant with GIL-18 gene was expressed and purified.Based on it,the polyclonal antibody of rabbit anti-rGIL-18 was made.

关 键 词：鹅 IL-18基因 克隆 原核表达 多克隆抗体 

分 类 号：Q511[生物学—生物化学] Q786