【摘要】 目的:构建TGFβ1基因RNAi慢病毒载体。方法:根据人TGFβ1的mRNA序列选择3个靶序列并根据它们设计合成3对寡核苷酸序列,同时合成1对阴性对照寡核苷酸序列;将以上4对寡核苷酸序列退火后连入pRNA-U6/Lenti质粒,酶切和测序鉴定后,将以上质粒分别和包装质粒混合物共转染293FT细胞,包装产生病毒颗粒。各组病毒载体转染Hela细胞后,运用Real-Time PCR和ELISA检测TGFβ1mRNA和蛋白的表达水平。结果:酶切和测序证实目的寡核苷酸片段已被准确克隆到pRNA-U6/Lenti质粒;各质粒与包装质粒共转染293FT细胞后能产生高滴度的慢病毒载体颗粒;各组慢病毒载体感染Hela细胞后,有两组可以显著抑制TGFβ1mRNA水平及蛋白水平的表达,其中以第一组效果最佳。结论:成功构建了人TGFβ1基因RNAi慢病毒载体。更多还原

【Abstract】 Objective: To construct a lentiviral vector of RNA interference(RNAi) of TGF-β1 gene. Methods: Three target sequences were selected according to TGFβ1 mRNA sequence firstly,then three pairs of oligonucleotide sequences according to these target sequences and one pair of negative control oligonucleotide sequence were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pRNA-U6/Lenti plasmid. After identified by restriction enzyme digestion and sequencing ,these plasmids were cotransfected along with ViraPowerTM Lentiviral Packaging Mix into 293FT cells for the package of lentiviral particles. Then the lentiviral vector particles were transfected into Hela cells and TGF-β1 expression in the transfected cells was assayed by Real-Time PCR and ELISA. Results: It was confirmed by digestion and sequencing that lentiviral vectors had the correct structure and could express high titer of virus. After transfected into Hela cells,TGF-β1 expression was decreased significantly by two of these lentiviral vectors at both the protein and mRNA level and the first vector had the best interefence efficiency. Conclusion: The lentivirus RNAi vector of TGF-β1 was constructed successfully.更多还原