绵羊ApoER2基因克隆及其干扰质粒对睾丸共培养细胞硒蛋白表达的影响

The Clone of ApoER2 Gene and the Inhibitory Effect of Interference Plasmid ApoER2-siRNA on Selenoprotein Expression in Co-culture Sertoli Cells of Sheep

本研究旨在克隆绵羊ApoER2基因,构建ApoER2-siRNA质粒载体,研究ApoER2基因沉默对睾丸共培养细胞SelP和PHGPx表达的影响。利用RT-PCR技术,克隆ApoER2序列,在此基础上构建4种pGPU6/GFP/Neo—ApoER2-siRNA质粒表达载体,利用脂质体将其转染到体外共培养绵羊睾丸细胞中,采用Real—time PCR和Western blotting方法,检测其抑制效应,并研究ApoER2基因沉默对Sel P和PHGPx mRNA表达的影响。本研究成功获得了长度为2 490 bp的绵羊睾丸ApoER2完整编码区序列,共编码892个氨基酸,绵羊ApoER2核苷酸序列与牛的同源性最高,相似性为97.7%。ApoER2-siRNA-1565和ApoER2-siRNA-2567位点重组质粒的干扰效果较好(P<0.01),选取ApoER2-siRNA-2567重组质粒,结果显示转染组Sel P和PHGPx表达量显著降低(P<0.01)。获得了绵羊ApoER2编码区序列和有效抑制该基因表达的2个质粒载体,ApoER2沉默可能影响了睾丸硒蛋白Sel P和PHGPx的表达,为进一步研究ApoER2转运硒机理提供了理论依据。

The aim of this study was to clone ovine ApoER2(apolipoprotein E receptor-2).construct the plasmid vector ApoER2-siRNA,and investigate the inhibitory effect of ApoER2 gene silencing on expression of Sel P and PHGPx genes in co-culture Sertoli cells of sheep.The ovine ApoER2 was cloned by RT-PCR.Four pGPU6/GFP/Neo-ApoER2 plasmid expression vectors were constructed,and transfected into the co-culture Sertoli cells of sheep by Lipofectamine2000?.The inhibitory effect of ApoER2-shRNA was detected by RT-PCR and Western blotting.The effect of ApoER2-shRNA inhibitory on expression of Sel P and PHGPx genes was detected by RT-PCR.The complete CDS of ApoER2 was 2 490 bp encoding a 892-amino-acid protein.The BLAST analysis showed that the entire nucleotide sequence of ApoER2 gene had 97.7%homology with bovine.ApoERZ mRNA levels in co-culture Sertoli cells were decreased significantly after transfection by the recombinant plasmids located in ApoER2-siRNA-1565(P<0.01) and ApoER2-siRNA-2567(P<0.01).Expression of Sel P and PHGPx mRNA were significantly decreased in the ApoER2-siRNA-2567 recombinant plasmid transfection group(P<0.01).The complete CDS of ApoER2 was obtained.Recombinant plasmid pGPU6/GFP/Neo-ApoER2-1565)and pGPU6/GFP/Neo-ApoER2-2567 were successfully constructed with effective inhibitory.Interference of ApoER2 gene decreased the expression of of Sel P and PHGPx mRNA,which could provide the scientific basis for further study of ApoER2 roles in pathway of selenium transport.