心脏发育候选基因PYGO1RNAi逆转录病毒载体系统的构建与鉴定

摘要

利用RNA干扰技术构建PYGO1基因RNA干扰(RNAi)的真核表达质粒(pSUPER -PYGO1).首先,针对 PYGO1 cDNA序列设计并化学合成一对编码短发夹RNA序列,经退火插入到由BglⅡ和XhoⅠ酶切的pSUPER 质粒上构建重组RNAi质粒pSUPER-PYGO1.通过XbaⅠ酶切鉴定及测序分析验证构建效果,将正确构建的质粒转染大鼠心肌细胞系H9c2建立产生逆转录病毒的细胞克隆.通过RT-PCR和Western blot检测pSUPER-PYGO1对H9c2细胞中PYGO1 mRNA和PYGO1蛋白的干扰效果.pSUPER-PYGO1质粒经酶切鉴定及测序分析,发现59nt寡核苷酸成功插入到预计位点,且序列完全一致;RT-PCR和Western blot检测结果显示转染 pSUPER-PYGO1的细胞中PYGO1 mRNA和PYGO1蛋白量明显降低.因此,靶向PYGO1的 pSUPER RNAi 载体构建成功,为进一步从分子水平探讨PYGO1在心脏发育中的功能奠定了基础.%The eukaryotic expression vector of pSUPER-PFGO1 that can inhibit PYGO1 gene expression was constructed. Firstly, a pair of oligo nucleotides coding for short hairpin RNA targeting gene PYGO1 were chemically synthesized, then annealed and inserted into pSUPER plasmids digested with Bgl Ⅱ and Xho I to construct the recombinant RNAi plasmid pSUPER-PYGO1. Recombinant pSUPER-PYGO1 plasmid was identified by enzyme digestion and sequencing a-nalysis. The H9c2 cells were transfected with the recombinant plasmid. The interferential effect was demonstrated by RT-PCR and Western blot. The result of enzyme digestion and sequencing analysis demonstrated that 59 nt oligonucleoti-des had been inserted successfully into the vector. RT-PCR and Western blot showed that pSUPER-PFGO1 can inhibit the transcription of PYGO1 mRNA and the expression of PYGO1 protein. The results of this studies implied that the pSU-PER RNAi vector targeting human heart developmental candidate gene PYGO1 were successfully constructed and it established a foundation for the study on the molecular function of PYGO1.