摘要
为了构建高效的靶向山羊PHGPx基因的干扰质粒,并在山羊共培养生精细胞中验证其抑制效应,利用Am-bion在线软件设计4对针对山羊PHGPx有潜在干扰效果的siRNA,体外退火后形成具有发夹结构的shRNA,将其与线性化的pGPU6/GFP/Neo载体相连形成质粒,然后经Pst I和BamH I酶切和测序鉴定;利用脂质体将其转染到体外共培养生精细胞中,用RT-PCR和Western印迹方法检测其PHGPx基因的抑制效应。结果表明:经Pst I和BamH I酶切和测序确定了150、182、214和248共4种pGPU6/GFP/Neo-PHGPx-shRNA的阳性克隆质粒;Lipo-fectamine 2000TM脂质体瞬时转染的共培养生精细胞的Real-time PCR结果表明,pGPU6/GFP/Neo-PHGPx-150、214和248质粒能够显著降低共培养细胞PHGPx mRNA的表达,蛋白免疫印迹结果图像分析免疫条带的平均光密度结果表明,pGPU6/GFP/Neo-PHGPx-214质粒极显著降低了共培养生精细胞PHGPx蛋白表达(P<0.01)。pG-PU6/GFP/Neo-PHGPx-shRNA质粒载体构建成功,pGPU6/GFP/Neo-PHGPx-214质粒瞬时转染共培养生精细胞后可以明显抑制PHGPx基因表达,可用于进一步PHGPx基因功能的研究。
The objectives of this study were to construct the specific interference plasmid targeting caprine PHGPx gene and identify the inhibitory effect on PHGPx expression in co-cultured spermtogenic cells of goats.Four siRNAs which had potential effect in interference were designed by using the online software in Ambion Company.Short hairpin siRNAs（shRNAs） were cloned into linearized pGPU6/GFP/Neo vector after annealing treatment siRNA in vitro.The pGPU6/GFP/Neo-PHGPx plasmid was identified by Pst I and BamH I restriction analysis and sequencing.The co-cultured spermtogenic cells were transfected by pGPU6/GFP/Neo-PHGPx with Lipofectamine 2000TM.The inhibitory effect of PHGPx gene was detected by RT-PCR and Western blotting.The four recombinant plasmids,pGPU6/GFP/Neo-PHGPx-150,182,214 and 248 were confirmed successfully by sequencing and Pst I and BamH I restriction analysise,then transiently transfected into the co-cultured spermatogenic cells.The PHGPx mRNA level in the co-cultured spermatogenic cells dramatically decreased after transfection by the recombinant plasmids pGPU6/GFP/Neo-PHGPx-150,214 and 248.The average optical density of western-blotting results showed that recombinant plasmid pGPU6/GFP/Neo-PHGPx-214 inhibited the expression of PHGPx protein significantly（P0.01）.The pGPU6/GFP/Neo-PHGPx-150,182,214 and 248 were successfully constructed.PHGPx gene was inhibited by recombinant plasmid pGPU6/GFP/Neo-PHGPx-214,which can be used in further study for PHGPx function.
出处
《山西农业大学学报（自然科学版）》
CAS
2012年第4期289-295,共7页
Journal of Shanxi Agricultural University(Natural Science Edition)
基金
国家自然科学基金资助项目(30871794)
