| 摘要: |
| 【目的】克隆猪细胞周期蛋白激酶9（CDK9）基因，观察该基因过表达对猪脐静脉血管内皮细胞（SUVEC）周期的影响。【方法】采用电子克隆方法筛选得到猪CDK9基因全长序列，并对其编码的蛋白进行生物信息学分析。利用RT-PCR方法从猪脾脏中扩增出包含完整开放读码框架(ORF)的CDK9 cDNA片段，将其与真核表达载体pEGFP-C1连接，构建重组真核表达载体pEGFP-CDK9。将pEGFP-CDK9以脂质体介导法转染至SUVEC细胞，采用绿色荧光标记和Western blot检测SUVEC细胞中CDK9基因的表达，用流式细胞仪检测转染CDK9基因后SUVEC细胞周期的变化。【结果】得到猪CDK9基因全长1 820 bp的cDNA序列，其ORF为1 119 bp，编码372个氨基酸；CDK9分子质量为42.76 ku，等电点为9.04；CDK9基因定位于猪的1号染色体上。酶切和测序结果证明，重组真核表达载体pEGFP-CDK9构建成功。pEGFP-CDK9转染SUVEC细胞48 h后,荧光显微镜和Western blot检测到目的基因序列在SUVEC细胞中成功表达；试验组各期SUVEC细胞比例与空载体组相比，差异无统计学意义（P＞0.05），细胞周期未发生显著变化。【结论】克隆了猪CDK9基因，并在SUVEC细胞中成功进行了表达。CDK9基因的过表达未引起细胞周期发生显著变化，其可能并不参与细胞周期的调控。 |
| 关键词: CDK9基因 猪 真核表达 细胞周期 |
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| 基金项目:西北农林科技大学引进人才基金项目(01140406)；国家自然科学基金项目(30270342) |
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| Cloning and effect of over-expression of cyclin-dependent kinase 9 on cell cycle of SUVEC cells |
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| Abstract: |
| 【Objective】CKD9 gene was cloned and the influence of CKD9 gene on the cell cycle of endothelial cell from swine umbilicus veins was observed.【Method】A CDK9gene was cloned by screening swine expressed sequence tag database,then it was analyzed via bioinformatics approaches.The complete open reading frame (ORF) of cDNA fragments was amplified from the swine spleen by RT-PCR.The swine CDK9 gene was cloned to the eukaryotic expression vector pEGFP-C1 and then transfected to SUVEC cell line by lipofectin.The expression of swine CDK9 was verified by GFP marker and Western blot.Cell cycle was analyzed by flow cytometry.【Result】The full length of CDK9 gene was 1 820 bp and its ORF 1 119 bp.It encoded a 372 amino acids protein with calculated molecular weight of 42.76 ku and isoelectric point of 9.04.And it was located on the chromosome1 of porcine genome.The recombinant plasmid containing the sequence of swine CDK9 gene was successfully constructed which was confirmed by restriction endonuclease digestion and sequencing.Our targeted gene was also successfully expressed on SUVEC 48 h post transfection and was co-confirmed by fluorescence microscope and western blot.Compared with the empty vector group,experimental group cells showed no significant change during each stage of cell cycle.【Conclusion】The swine CDK9gene was cloned and expressed effectively on SUVEC.Its over expression did not cause significant changes in cell cycle,which seemed that no detectable relationship existed between CDK9 and cell cycle regulation. |
| Key words: CDK9 gene swine eukaryotic expression cell cycle |
