靶向人Ⅰ型胶原蛋白α1链分子的shRNA真核表达载体的构建与鉴定  

Construction and identification of the eukaryotic expression vector expressing short hairpin RNA targeting human COL1A1

作　　者：余海浪[1] 周珏宇[1] 孟伟[1] 郑文岭[2] 马文丽[1] 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515 [2]华南基因组中心,广东广州510800

出　　处：《细胞与分子免疫学杂志》2013年第10期1032-1035,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：广东省医学科研基金(B2012203);广东省领军人才专项基金(C1030925)

摘　　要：目的构建靶向人Ⅰ型胶原蛋白α1链(COL1A1)分子的shRNA真核表达载体并检测其对靶分子的干扰效应。方法根据靶分子COL1A1基因序列,设计、合成编码其shRNA的互补寡核苷酸序列,克隆至线性化的pSilencerTM2.1-U6 neo载体中并对重组质粒进行酶切分析及测序鉴定;脂质体介导重组质粒转染乳腺癌MDA-MB-231细胞,经G418筛选获稳定表达细胞株,RT-PCR及Western blot法检测其对靶分子COL1A1表达的影响。结果酶切及DNA测序证实重组质粒pshRNA-COL1A1序列正确。与对照组比较,重组表达质粒pshRNA-COL1A1-1和pshRNA-COL1A1-2在mRNA和蛋白水平均能显著抑制COL1A1基因的表达(P<0.05),抑制率分别为(44.41%±3.90%,63.05%±3.13%)及(45.50%±2.71%,66.98%±2.08%)。结论成功构建靶向人乳腺癌MDA-MB-231细胞COL1A1基因的shRNA真核表达载体。Objective To construct the eukaryotic expression vector of short hairpin RNA （shRNA） targeting human collagen type 1 alpha 1 （COL1A1） and observe its inhibiting effect on the expression of target gene. Methods The complementary oligonucleotide sequences coding shRNA were designed and synthesized according to the sequence of human COL1A1 gene, and cloned into the linearized pSilencerTM2. I-U6 neo vector. The recombinant vector was confirmed by enzyme digestion analysis and DNA sequencing, and then the positive clones were transfected to human breast cancer cell MDA-MB-231 by LipofectamineTM2000. The stable cell line was selected by G418. The expression of COL1A1 gene was detected by semi-quantitative RT-PCR and Western blot analysis. Results Double-enzyme digestion and DNA sequencing verified the correct sequences of the recombinant plasmid pshRNA-COL1A1. Compared with the control group, the expression level of COLIA1 mRNA and protein was inhibited markedly by pshRNA-COL1A1-1 or pshRNA-COL1A1-2 transfection, and the inhibitory rates were respectively （44.41± 3.90）%, （63.05±3.13）% in RT-PCR and （45.50 ±2.71）%, （66.98±2.08）% in Western blot analysis. Conclusion Specific shRNA interference plasmid vector targeting COL1A1 gene mRNA was constructed successfully.

关 键 词：乳腺癌 I型胶原蛋白d1链 短发夹RNA 真核表达载体 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]