RNA干涉用于Survivin性质的研究
发布时间：2018-11-28 17:56
【摘要】：Survivin 是1997 年Ambrosini 等应用EPR-1 基因cDNA 作为探针去筛选人基因组文库而得到的,是IAP 家族的一个成员,以其独特的分子结构以及表达特点,在其被发现后的几年中得到广泛的研究与关注。 本论文中,我们用蛋白合成抑制剂放线菌酮CHX处理宫颈癌细胞HeLaS3,并用Western-blot 检测,发现60min 后Survivin 蛋白的表达量明显降低。而用PKA 的抑制剂H89 预先处理细胞两个小时后,再用CHX 处理细胞,则Survivin蛋白量变化不明显。我们推断,Survivin 的稳定性可能与其潜在的PKA 磷酸化位点的磷酸化状态有关。 进而通过序列比对,我们选择出三条针对Survivin 基因的siRNA 片段,并构建了相应的RNAi 载体,转染入HeLaS3 细胞后,瞬时转染的结果表明,Survivin 表达量在RNA 和蛋白水平上都得以下调,尤以S3 片段效果最为明显,并且用流式细胞术检测干涉载体转染对细胞产生的生物学效应,发现转染干涉载体pTet-U6-S3 后,细胞凋亡率得到很大的提高。 另外,我们构建了Survivin 上潜在的pKA 磷酸化位点Ser81 的突变体S81D、S81A,用来检测该位点磷酸化不同状态对Survivin 稳定性的影响。 但是把干涉载体转染至细胞,并把其培养成稳定株后,HeLaS3 细胞内源Survivin 量并没有被有效地抑制,这是很出乎预料的。
[Abstract]:Survivin was obtained from 1997 when Ambrosini used EPR-1 gene cDNA as a probe to screen human genome library. It is a member of IAP family with its unique molecular structure and expression characteristics. In the years since its discovery, it has received extensive research and attention. In this paper, we treated cervical cancer cell HeLaS3, with actinomycin CHX, a protein synthesis inhibitor, and detected by Western-blot. It was found that the expression of Survivin protein decreased significantly after 60min. However, when the cells were pretreated with H89, a PKA inhibitor, and then treated with CHX for two hours, the Survivin protein content did not change significantly. We infer that the stability of Survivin may be related to the phosphorylation state of its potential PKA phosphorylation site. Then through sequence alignment, we selected three siRNA fragments targeting Survivin gene and constructed corresponding RNAi vector. After transfection into HeLaS3 cells, the transient transfection results showed that the expression of Survivin was down-regulated at both RNA and protein levels. The effect of S3 fragment was the most obvious, and the biological effect of interference vector transfection on cells was detected by flow cytometry. It was found that after transfection of interference vector pTet-U6-S3, cell apoptosis rate was greatly increased. In addition, we constructed the mutant S81D- S81A of Ser81, a potential pKA phosphorylation site on Survivin, to detect the effect of different phosphorylation states on the stability of Survivin. However, after the interference vector was transfected into the cells and cultured into a stable strain, the amount of endogenous Survivin in HeLaS3 cells was not effectively inhibited, which was unexpected.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
本文编号：2363775
[Abstract]:Survivin was obtained from 1997 when Ambrosini used EPR-1 gene cDNA as a probe to screen human genome library. It is a member of IAP family with its unique molecular structure and expression characteristics. In the years since its discovery, it has received extensive research and attention. In this paper, we treated cervical cancer cell HeLaS3, with actinomycin CHX, a protein synthesis inhibitor, and detected by Western-blot. It was found that the expression of Survivin protein decreased significantly after 60min. However, when the cells were pretreated with H89, a PKA inhibitor, and then treated with CHX for two hours, the Survivin protein content did not change significantly. We infer that the stability of Survivin may be related to the phosphorylation state of its potential PKA phosphorylation site. Then through sequence alignment, we selected three siRNA fragments targeting Survivin gene and constructed corresponding RNAi vector. After transfection into HeLaS3 cells, the transient transfection results showed that the expression of Survivin was down-regulated at both RNA and protein levels. The effect of S3 fragment was the most obvious, and the biological effect of interference vector transfection on cells was detected by flow cytometry. It was found that after transfection of interference vector pTet-U6-S3, cell apoptosis rate was greatly increased. In addition, we constructed the mutant S81D- S81A of Ser81, a potential pKA phosphorylation site on Survivin, to detect the effect of different phosphorylation states on the stability of Survivin. However, after the interference vector was transfected into the cells and cultured into a stable strain, the amount of endogenous Survivin in HeLaS3 cells was not effectively inhibited, which was unexpected.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
【参考文献】
相关期刊论文 前1条
1 吴峰,陈惠鹏;通过RNA干涉沉默SARS病毒的研究前景[J];生物技术通讯;2003年03期
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