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目的:构建pAAV-NF-YA真核表达载体并转染子宫颈癌HeLa细胞,检测NF-YA的表达水平。方法:抽提人类胚胎干细胞和胚胎成纤维细胞的总RNA,RT-PCR获得NF-YA基因的cDNA,克隆至pAAV-MCS载体中,经限制性酶切和测序鉴定后,重组质粒pAAV-NF-YA转染HeLa细胞,Western Blot检测NF-YA的表达。结果:成功构建了NF-YA的真核表达载体,转染HeLa细胞后NF-YA的表达水平有显著提高。结论:获得了有效的pAAV-NF-YA真核表达载体,为后续NF-Y的功能研究提供了便利。
Objective: To construct pAAV-NF-YA eukaryotic expression vector and transfect cervical cancer HeLa cells to detect the expression of NF-YA. Methods: The total RNA of human embryonic stem cells and embryonic fibroblasts was extracted. The cDNA of NF-YA gene was obtained by RT-PCR and cloned into pAAV-MCS vector. After restriction enzyme digestion and sequencing, the recombinant plasmid pAAV-NF -YA transfected HeLa cells, Western Blot detection of NF-YA expression. Results: The eukaryotic expression vector of NF-YA was successfully constructed. The expression of NF-YA in HeLa cells was significantly increased after transfection. Conclusion: The pAAV-NF-YA eukaryotic expression vector was obtained, which provided convenience for the subsequent study of the function of NF-Y.