靶向人类心脏发育候选基因hole的siRNA真核表达载体的构建和鉴定  

Construction and Identification of Eukaryotic siRNA Expression Vector Targeting Human Heart Developmental Candidate Gene hole Gene

作　　者：周军媚[1,2,3] 姚峰[4] 谢华平[2,3] 叶湘漓[2,3] 王跃群[2,3] 

机构地区：[1]南华大学药学与生命科学学院,湖南省衡阳市421001 [2]南华大学 [3]湖南师范大学生命科学学院,湖南省长沙市410006 [4]南华大学实验动物学部,湖南省衡阳市421001

出　　处：《中国动脉硬化杂志》2012年第5期397-401,共5页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81000035;31071999和30871340)

摘　　要：目的利用siRNA表达载体构建靶向人类心脏发育候选基因hole的pSUPER RNAi载体系(pSUPER-hole)及筛选稳定产毒的细胞克隆。方法化学合成一对编码短发夹RNA序列的、靶向人类心脏发育候选基因hole的寡核苷酸链60个碱基,退火,克隆到经BglⅡ、XhoⅠ双酶切的pSUPER质粒上,构建重组RNAi质粒(pSU-PER-hole)。通过双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H9c2,经过puro抗生素的筛选,建立稳定产生逆转录病毒的细胞模型,采用RT-PCR检测其抑制效果。结果 pSUPER-hole载体经双酶切鉴定及测序分析,结果表明60个碱基成功插入到预计位点,并且序列完全一致。重组载体转染包装细胞,筛选的细胞克隆均可表达绿色荧光蛋白,表明包装成功。RT-PCR检测表明pSUPER-hole能成功抑制hole基因的表达。结论靶向人类心脏发育候选基因hole的siRNA真核表达载体构建成功。Aim To construct a pSUPER RNAi system that targets human heart developmental candidate gene hole and a stable virus-producing cell line. Methods A pair of 60nt oligonucleotides coding for short hairpin RNA and targeting human heart developmental candidate gene hole were chemically synthesized and annealed and inserted into pSUPER plasmids digested with Bgl II and Xho I to construct the recombinant pSUPER RNAi plasmid （pSUPER-hole）. Recombinant pSUPER-hole plasmid was identified by enzyme digestion and sequencing analysis. The packaging cell H9c2 was transfected with the recombinant plasmid. The mRNA expression level of hole was detected by RT-PCR. Results The result of enzyme digestion and sequencing analysis demonstrated that 60 nt had been inserted successfully into the vector. Green fluorescent was detected in virus-producing cells, RT-PCR showed pSUPER-hole can inhibit the hole gene expression of H9c2. Conclusion The pSUPER RNAi vector targeting human heart developmental candidate gene hole are successfully constructed.

关 键 词：心脏发育 HOLE PSUPER siRNA H9C2细胞 

分 类 号：Q34[生物学—遗传学]