心脏发育候选基因PYGO1 RNAi逆转录病毒载体系统的构建与鉴定  

Construction and Identification of RNAi Recombinant Retroviral Vectors Expression siRNA for Heart Developmental Candidate Gene PYGO1

作　　者：刘明[1] 刘宪楚[1] 周军媚[1,2] 谢华平[1] 廖四芳[1] 宋文[1] 李佑锋[1] 吴秀山[1] 王跃群[1] 

机构地区：[1]湖南师范大学生命科学学院心脏发育研究中心,湖南长沙410006 [2]南华大学药学与生命科学学院,湖南衡阳421001

出　　处：《激光生物学报》2012年第6期528-531,540,共5页Acta Laser Biology Sinica

基　　金：国家自然科学基金(30930054;31071999;31272396)

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pSU-PER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pSU-PER-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-PYGO1 that can inhibit PYGO1 gene expression was construc- ted. 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 ceils 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-PYGO1 can inhibitthe 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 estab- lished a foundation for the study on the molecular function of PYGO1.

关 键 词：PYGO1基因 RNA干扰 PSUPER H9C2 

分 类 号：Q78[生物学—分子生物学]