内江猪γ干扰素基因的克隆、序列分析及真核表达载体的构建  

Molecular Cloning,Sequencing and Constructing Eukaryotic Expression Vector of Neijiang Pig IFN-γ

作　　者：黄亚平[1] 郭万柱[1] 李晓琪[1] 

机构地区：[1]四川农业大学动物生物技术中心,雅安625014

出　　处：《中国畜牧兽医》2007年第5期60-63,共4页China Animal Husbandry & Veterinary Medicine

基　　金：国家"973"项目(2004CCA01800)

摘　　要：将内江猪的外周血淋巴细胞在刀豆素(ConA)的刺激下培养24 h后,提取总RNA,应用一步法RT-PCR扩增γ干扰素(IFN-γ)cDNA,克隆到PMD18-T载体,命名为pMD-N-IFN-γ,测序结果证明克隆的cDNA全长603 bp,其ORF为501bp,编码166个氨基酸,与Genbank公布的IFN-γ比对,核苷酸同源性在99.4%以上,从而证实成功地克隆了内江猪IFN-γ基因。以pMD-N-IFN-γ质粒为模板亚克隆完整ORF区,连接到真核表达质粒pcDNA3.1(+)的EcoRI、XhoI两位点之间,经单双酶切鉴定,成功的构建了IFN-γ真核表达载体pcDNA3.1(+)-N-IFN-γs。Total RNA was isolated from neijiang pig peripheral blood lymphocytes which were stimulated with ConA. Then the pIFN-γ cDNA was amplified by reverse transcription polymerase chain reaction. The amplified fragment was cloned into vector pMD18-T named pMD-N-IFN-γ and then sequenced. It was found that the amplified fragment was consisted of 603 nucleotides, the ORF consisted of 501 nucleotides, encoding 166 amino acids, Neijang pig IFN-γ gene has more than 99.4% homology with other pigs on Genbank. Sub-cloning the ORF of pig IFN-γ gene, then the sub-cloned gene was successfully inserted between EcoRI and XhoI site of the eukaryotic expression vector pcDNA3.1 （＋）, named pcDNA3. 1（＋）-N-IFN-γs.

关 键 词：Γ干扰素 克隆 序列分析 真核表达载体构建 

分 类 号：Q785[生物学—分子生物学]