绵羊itgαv基因靶向RNAi表达载体的构建和筛选

Construction and screening of sheep itgαv gene targeting RNAi expression vector

为了探讨和分析整联蛋白αv亚基基因(integrin alpha v,itgαv基因)的功能及受体基因表达量下调对口蹄疫病毒复制的影响,试验构建并筛选了绵羊itgαv基因的RNAi载体,根据绵羊itgαv基因核苷酸序列,设计了3条特异性双链小干扰RNA(short interfering RNA,siRNA)分子,将合成的DNA片段退火形成双链后,连接到p Genesil-1.3载体人源U6启动子下游,分别命名为Genesil-αv1、Genesil-αv2、Genesil-αv3;以乳腺组织RNA反转录产物为模板,扩增绵羊itgαv,并将其克隆入p EGFP-N1载体多克隆位点,构建itgαv与GFP蛋白融合表达载体p EGFP-itgαv,用上述Genesil-αv1、Genesil-αv2、Genesil-αv3载体分别与p EGFP-itgαv共转染豚鼠BHK-21细胞之后,通过绿色荧光信号观察和半定量RT-PCR检测siRNA分子对itgαv基因表达的抑制效果,经酶切及测序鉴定以及瞬转试验证实,载体表达的RNAi分子构建成功,且能有效抑制目标基因的体外表达。结果表明:研究成功构建了有效抑制itgαv基因表达的RNAi载体。

To investigate and analyze the function of integrin alpha v （itgctv） gene and the down - regulated expression level of the receptor gene on the influence of the foot - and - mouth disease virus replication, the RNA interference （RNAi） vector of sheep itgav gene was constructed and screened. The three specific double - stranded short interfering RNA interference （siRNA） molecules were designed according to the nucle-otide sequence of sheep itgav gene. After the synthetic DNA fragments annealed to form double - stranded, the fragments were connected to the downstream of humanized U6 promoter for vector pGenesil - 1.3, were named as Genesil - avl, Genesil -av2, and Genesil - av3, respectively. The sheep itgav gene was amplified using the reverse transcribed product of mammary tissue RNA, and then was cloned into the multiple cloning sites of vector pEGFP - N1 to build itgetv with GFP fusion protein expression vector pEGFP - itgav. After the above vectors including Genesil - avl, Genesil - av2 and Genesil - av3 were used to co - transfect the guinea pig BHK - 21 cells with pEGFP - itgav, the inhibiting effects of siRNA molecules on the itgav gene expression were detected through green fluorescence sigual observation and semi - quantitative RT - PCR assay. The siRNA molecules with vector expression were successfully constructed and could effectively inhibit the in vitro expression of the target gene, which was confirmed by enzyme digestion and sequencing identification and instantaneous turning test. This study successfully constructs the RNAi vector to effec/ively inhibit the itgav gene expression, and lays a foundation for the study of the deletion model cells of sheep itgav gene.