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

发布时间：2019-02-27 15:52

【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(P0.05),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。
[Abstract]:Aim to construct the eukaryotic expression vector of shRNA targeting human type I collagen 伪 1 chain (COL1A1) and detect its interference effect on the target molecule. Methods according to the target molecule COL1A1 gene sequence, the complementary oligonucleotide sequence encoding the shRNA was synthesized and cloned into the linearized pSilencerTM2.1-U6 neo vector. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into breast cancer cell line MDA-MB-231 by lipofectamine, and the stable expression cell line was screened by G418. The effect of the recombinant plasmid on the expression of target molecule COL1A1 was detected by RT-PCR and Western blot. Results restriction endonuclease digestion and DNA sequencing confirmed that the recombinant plasmid pshRNA-COL1A1 was correct. Compared with the control group, the recombinant expression plasmids pshRNA-COL1A1-1 and pshRNA-COL1A1-2 could significantly inhibit the expression of COL1A1 gene at the mRNA and protein levels (P0.05), and the inhibition rates were (44.41% 卤3.90%), respectively. (63.05% 卤3.13%) and (45.50% 卤2.71%, 66.98% 卤2.08%). Conclusion Eukaryotic expression vector shRNA targeting COL1A1 gene in human breast cancer MDA-MB-231 cells was successfully constructed.
【作者单位】：南方医科大学基因工程研究所;华南基因组中心;
【基金】：广东省医学科研基金(B2012203) 广东省领军人才专项基金(C1030925)
【分类号】：R346

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