靶向FMDV受体猪源整联蛋白α_V亚基基因抑制FMDV复制的最佳siRNA筛选  

The Optimum siRNA Screening of Targeting Porcine Integrin α_v Subunit Gene as FMDV Receptor Against FMDV Replication

作　　者：骆继怀[1] 独军政[1] 高闪电[1] 张国锋[1] 常惠芸[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国家口蹄疫参考实验室甘肃省生物检测工程技术研究中心,兰州730046

出　　处：《畜牧兽医学报》2011年第12期1732-1737,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：农业部转基因重大专项(2009ZX08007-008B;2009ZX08006-002B;2008ZX08011-004)

摘　　要：筛选靶向FMDV受体猪源整联蛋白αV亚基基因抑制FMDV复制的最佳siRNA。根据猪源αV mRNA序列,设计并合成siRNA,Lipofectamine 2000转染siRNA于PK-15细胞,利用qRT-PCR检测RNAi组(iαV-480、iαV-1719和iαV-2077)、空白组(Mock)和阴性对照组(Control)中αVmRNA表达情况;在转染siRNA12h后通过接种100TCID50,收集病毒液,测定TCID50,确定其抗病性变化。结果显示,瞬间转染PK-15后,与阴性对照组和空白组相比,3个RNAi组均不同程度抑制αV亚基基因表达,在24hiαV-480组在mRNA水平抑制90.1%,作用最明显。TCID50测定表明iαV-480组有较低的病毒滴度,说明其抗病性增加。针对猪源整联蛋白αV亚基基因的最佳siRNA的筛选成功,为深入研究干扰FMDV受体抗FMDV转基因路线奠定了基础。To screen the optimum siRNA targeting porcine integrin αV subunit as FMDV receptor against FMDV replication,three siRNA sequences were selected according to porcine integrin αV mRNA sequence,the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized.After PK-15 cells were transected with siRNAs by Lipofectamine 2000,qRT-PCR and 100 TCID50 were used to evaluate the level of αV expression and resistance of FMDV O/CHA/99,respectively.The results showed that compared with control and mock group,the αV subunit mRNA expression were obviously suppressed in all three experimental groups（iαV-480,iαV-1719 and iαV-2077）,especially the expression rate in iαV-480 group was reduced by 90.1% in 24 hours.Lower virus titers of iαV-480 group by TCID50 indicated that PK-15 cells increased the resistance to FMDV O/CHA/99.The optimum siRNA toward porcine integrin αV subunit gene are successfully screened in this study,it would be helpful work about studying of interference FMDV receptor gene anti-FMD transgene in swine.

关 键 词：FMDV siRNA 转染 PK-15细胞 αv亚基基因 病毒滴度 

分 类 号：S852.659.6[农业科学—基础兽医学]