AE3基因siRNA表达质粒的构建及对其转染的心肌细胞H9c2的影响
发布时间：2018-11-04 14:34
【摘要】： 目的:利用RNA干扰(RNAi)技术,以AE3为靶基因,设计构建siRNA表达质粒,进行测序鉴定,并检测该表达质粒对大鼠心肌样细胞系H9c2 AE3基因表达的影响,为进一步研究AE3基因在心肌细胞损伤及保护中的作用奠定基础。 方法: 1.设计具有短发夹结构的三条DNA序列,经退火成互补双链,再克隆至载体pSilencer3.1-H1-hygro中构建重组表达载体,转化DH5α菌株,提取质粒并进行序列测定。 2.转染携带绿色荧光蛋白的pEGFP-N2质粒,荧光显微镜观察检测瞬时转染不同时相的转染效率,选择最佳时相用于后续瞬时转染靶基因表达抑制率的分析。 3.利用构建成功的siRNA表达质粒,通过脂质体介导转染H9c2心肌样细胞,并通过RT-PCR、Western blot检测细胞中AE3 mRNA和蛋白的表达。 结果: 1.重组质粒转化DH5α菌株经氨苄青霉素抗性筛选可见有细菌克隆长出。 2.测序鉴定表明重组质粒中含有针对AE3基因的目的序列。 3.将pEGFP-N2通过脂质体介导,转染H9c2心肌样细胞,分别于转染24 h、48 h、72 h后在荧光显微镜下观察转染效果,可见H9c2心肌样细胞胞浆内有绿色荧光,其中转染48 h的转染效率82±6%显著高于转染24 h的转染效率43±6% (P0.01)和转染72 h的转染效率60±7% (P0.05)。因此,按48 h最佳转染时间转染构建好的siRNA表达质粒。 4. RT-PCR检测细胞中AE3 mRNA的表达水平,显示pSilencer-AE3-A可明显降低H9c2心肌样细胞中AE3 mRNA的表达。 5. Western blot检测细胞中AE3蛋白的表达量,显示pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C转染的H9c2心肌样细胞AE3蛋白表达分别降低61%,48%,25%。 结论: 1.成功构建AE3基因siRNA表达质粒pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C。 2.重组质粒pSilencer-AE3-A可明显降低H9c2心肌样细胞AE3基因在mRNA和蛋白水平的表达。
[Abstract]:Objective: to design and construct siRNA expression plasmid using AE3 as target gene by RNA interference (RNAi) technique, and to detect the effect of siRNA expression plasmid on the expression of H9c2 AE3 gene in rat cardiomyoid cell line. It provides a basis for further study of the role of AE3 gene in myocardial injury and protection. Methods: 1. Three DNA sequences with short hairpin structure were designed and annealed into complementary double strands, then cloned into vector pSilencer3.1-H1-hygro to construct recombinant expression vector. The recombinant expression vector was transformed into DH5 伪 strain. The plasmid was extracted and sequenced. 2. After transfection of pEGFP-N2 plasmid carrying green fluorescent protein, the transfection efficiency of different phases of transient transfection was observed by fluorescence microscope, and the optimal phase was selected to analyze the inhibition rate of target gene expression in subsequent transient transfection. 3. The siRNA expression plasmid was constructed and transfected into H9c2 cardiomyoid cells by liposome. The expression of AE3 mRNA and protein was detected by RT-PCR,Western blot. Results: 1. The recombinant plasmid transformed DH5 伪 strain was screened for ampicillin resistance. 2. Sequencing analysis showed that the recombinant plasmid contained the target sequence for AE3 gene. 3. PEGFP-N2 was transfected into H9c2 cardiomyocytes via liposome, and the transfection effect was observed under fluorescence microscope at 24 h, 48 h or 72 h after transfection. Green fluorescence was observed in the cytoplasm of H9c2 cardiomyoid cells. The transfection efficiency of 48 h was significantly higher than that of 24 h (P 0.01) and 72 h (P 0.05). Therefore, the siRNA expression plasmid was constructed at the best transfection time of 48 h. 4. The expression of AE3 mRNA was detected by RT-PCR, which showed that pSilencer-AE3-A could significantly decrease the expression of AE3 mRNA in H9c2 cardiomyocytes. 5. The expression of AE3 protein was detected by Western blot. The results showed that the expression of AE3 protein in H9c2 cardiomyocytes transfected with pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C decreased by 61% and 25% respectively. Conclusion: 1. Construction of siRNA expression plasmid pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C. of AE3 gene 2. Recombinant plasmid pSilencer-AE3-A could significantly reduce the expression of AE3 gene in H9c2 cardiomyocytes at the level of mRNA and protein.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R541;R346
[Abstract]:Objective: to design and construct siRNA expression plasmid using AE3 as target gene by RNA interference (RNAi) technique, and to detect the effect of siRNA expression plasmid on the expression of H9c2 AE3 gene in rat cardiomyoid cell line. It provides a basis for further study of the role of AE3 gene in myocardial injury and protection. Methods: 1. Three DNA sequences with short hairpin structure were designed and annealed into complementary double strands, then cloned into vector pSilencer3.1-H1-hygro to construct recombinant expression vector. The recombinant expression vector was transformed into DH5 伪 strain. The plasmid was extracted and sequenced. 2. After transfection of pEGFP-N2 plasmid carrying green fluorescent protein, the transfection efficiency of different phases of transient transfection was observed by fluorescence microscope, and the optimal phase was selected to analyze the inhibition rate of target gene expression in subsequent transient transfection. 3. The siRNA expression plasmid was constructed and transfected into H9c2 cardiomyoid cells by liposome. The expression of AE3 mRNA and protein was detected by RT-PCR,Western blot. Results: 1. The recombinant plasmid transformed DH5 伪 strain was screened for ampicillin resistance. 2. Sequencing analysis showed that the recombinant plasmid contained the target sequence for AE3 gene. 3. PEGFP-N2 was transfected into H9c2 cardiomyocytes via liposome, and the transfection effect was observed under fluorescence microscope at 24 h, 48 h or 72 h after transfection. Green fluorescence was observed in the cytoplasm of H9c2 cardiomyoid cells. The transfection efficiency of 48 h was significantly higher than that of 24 h (P 0.01) and 72 h (P 0.05). Therefore, the siRNA expression plasmid was constructed at the best transfection time of 48 h. 4. The expression of AE3 mRNA was detected by RT-PCR, which showed that pSilencer-AE3-A could significantly decrease the expression of AE3 mRNA in H9c2 cardiomyocytes. 5. The expression of AE3 protein was detected by Western blot. The results showed that the expression of AE3 protein in H9c2 cardiomyocytes transfected with pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C decreased by 61% and 25% respectively. Conclusion: 1. Construction of siRNA expression plasmid pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C. of AE3 gene 2. Recombinant plasmid pSilencer-AE3-A could significantly reduce the expression of AE3 gene in H9c2 cardiomyocytes at the level of mRNA and protein.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R541;R346
【共引文献】
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