稳定表达外源TLR9基因的山羊胎儿成纤维细胞系的建立  

Establishment of the goat fetal fibroblast cell lines stably expressing exogenous TLR9 gene

作　　者：刘太安[1] 房永祥[1] 贾怀杰[1] 马保华[2] 何小兵[1] 管齐赛[1] 陈国华[1] 曾爽[1] 姚文娟[1] 景志忠[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点实验室,甘肃兰州730046 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医科学》2013年第6期605-611,共7页Chinese Veterinary Science

基　　金：转基因生物新品种培育重大专项(2009ZX08008-010B);国家自然科学基金资助项目(30871884)

摘　　要：为提供制备抗病转基因山羊核供体细胞,研究Toll样受体9(Toll like receptor 9,TLR9)对山羊抗病性能的影响,应用反转录聚合酶链式反应(RT-PCR)技术,从山羊外周血单核细胞中克隆了TLR9基因,并对其遗传进化关系及分子结构特征进行了分析,发现克隆的基因具有Toll样受体家族的典型结构特征,其遗传进化关系与物种亲缘关系密切相关。将克隆的山羊TLR9基因亚克隆至pIRES2-EGFP真核表达载体,转染山羊胎儿成纤维细胞,经G418筛选以及阳性细胞基因组PCR鉴定和荧光观察证实,外源TLR9基因确已稳定整合入转染阳性细胞克隆基因组中并获得表达。成功建立了稳定表达外源TLR9的山羊胎儿成纤维细胞系,为培育抗病转基因山羊奠定了基础。To provide the nuclear donor cell lines for the production of anti-disease transgenic goats and to study the influence of Toll-like receptor 9（TLR9） on the disease-resistant ability of goat,the coding sequence of goat TLR9 gene was amplified from peripheral blood mononuclear cells by reverse transcriptionpolymerase chain reaction. Furthermore, the genetic evolution and molecular structure of the goat TLR9 gene were analyzed, and the results indicated that this sequence shared much typical characteristics with Toll-like receptor sequences and was rather relative to the species evolution. Subsequently, the cloned TLR9 gene was subcloned into the eukaryotic expression vector pIRES2-EGFP. The recombinant plasmid was then transfected into the goat fetal fibroblasts. Through G418 selection, PCR and fluorescence detection,exogenous TLR9 gene was demonstrated to be integrated into the genomes of the positive cell clones and the cell clones stably expressed exogenous TLR9. In conclusion, the fibroblast cell lines stably expressing exogenous TLR9 were established,which will provide foundation for breeding of anti-disease transgenic goats.

关 键 词：TLR9基因 转基因山羊 山羊胎儿成纤维细胞 稳定表达 

分 类 号：R979.5[医药卫生—药品]