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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 shRNA eukaryotic expression vector targeting human COL1A1 molecule and test its interference effect on target molecules. Methods According to the COL1A1 gene sequence of target, a complementary oligonucleotide sequence encoding its shRNA was designed and synthesized and cloned into the linearized pSilencerTM2.1-U6 neo vector. The recombinant plasmid was digested and sequenced. Liposome The recombinant plasmids were transfected into breast cancer MDA-MB-231 cells. The stable cell lines were screened by G418. The expression of COL1A1 was detected by RT-PCR and Western blot. Results Restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pshRNA-COL1A1 sequence was correct. Compared with the control group, the expression of pshRNA-COL1A1-1 and pshRNA-COL1A1-2 at the mRNA and protein levels significantly inhibited COL1A1 expression (P <0.05), and the inhibitory rates were (44.41% ± 3.90%, 63.05 % ± 3.13%) and (45.50% ± 2.71%, 66.98% ± 2.08%). Conclusion The shRNA eukaryotic expression vector targeting COL1A1 gene of human breast cancer MDA-MB-231 cells was successfully constructed.