兰州大学机构库 >生命科学学院
Fkbp19基因的初步研究及抗体制备
Alternative TitleThe primary research of Fkbp19 and the preparation of antibody
范开吉
Thesis Advisor刘迺发 ; 杨晓
2010-05-17
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordFkbp19 肝细胞癌 蛋白合成分泌 RNA干扰 生长曲线 细胞周期 Transwell 多克隆抗体
Abstract我们利用芯片筛选的方式首先发现Fkbp19在HBx-p21转基因小鼠的HCC组织中是高表达的,并用Northern blot的方法进行了验证;而且在病人HCC组织中检测Fkbp19的表达发现,有75%的病例显示Fkbp19在HCC组织的表达量要高于正常组织。这提示Fkbp19的异常高表达与HCC的发生发展有着较高的相关性。 回顾文献我们发现,Fkbp19是一个功能未知基因,目前仅有一篇文献报道它是FKBP家族的一个成员并具有就较弱的FK506结合能力。通过对Fkbp19的基因和蛋白结构分析我们发现它有可能定位于内质网及核仁上。Northern blot 检测显示其在多种外分泌腺中有表达,亚细胞定位研究发现其可以定位于内质网、核仁及线粒体上。这些结果提示Fkbp19与蛋白的合成分泌密切相关。 接下来我们使用RNAi的技术来敲低Fkbp19的表达并检测相关细胞表型的变化。我们选择了Fkbp19表达量最高的乳腺癌细胞系ZR75-1来进行此次干涉实验,经过成功的干涉载体构建,质粒的稳定转染筛选成功得到了稳定干涉载体的细胞。两种不同敲低效率的检测显示我们得到了Fkbp19敲低的细胞,可以进行下步实验。 我们利用MTT的方法来检测Fkbp19敲低后细胞生长能力的变化,结果发现细胞生长明显减慢,而对细胞周期(FCM)检测结果表明细胞发生了G1期阻滞,相应的推动细胞经过G1/S检查点的蛋白CDK4及CCND1的表达也显著下降。Transwell实验证明细胞在敲低Fkbp19表达后细胞的迁移能力明显下降。 由于市面上没有商品化的Fkbp19的抗体,我们就自行制备了Fkbp19的兔源多克隆抗体:经过IPTG诱导的原核表达,基于His标签的蛋白纯化,免疫家兔,收集血清,我们的得到的抗血清可以很好的用于Western blot检测内源以及外源表达的Fkbp19蛋白。 总之,我们发现Fkbp19的表达在小鼠和人的HCC组织中是升高的,在外分泌腺高表达并与蛋白的合成分泌关系密切。敲低Fkbp19的表达使细胞的生长迁移的能力减弱,提示Fkbp19高表达对于肿瘤的发生发展又促进作用。另外我们成功制备了兔源Fkbp19多克隆抗体,以便对Fkbp19的蛋白水平进行检测,为进一步阐明Fkbp19的功能打下基础。
Other AbstractThrough microarray analysis, Fkbp19 was identified to be upregulated in HCC tissues from the HBX-p21 transgenic mouse, which was confirmed by using the northern blot technology. The expression level of Fkbp19 in the tissues of HCC from the patients was also analyzed and the result showed that the expression of Fkbp19 was higher in 75% of the subjects’ HCC tissue than adjacent non-cancerous tissues, which showed high correlation between the aberrant expression of Fkbp19 and the HCC. The Fkbp19 was a function-unknown gene, it was only reported that Fkbp19 was one member with low FK506 binding activity of FKBP protein family. Sequence analysis indicated that Fkbp19 protein might be an endoplasmic reticulum- and nucleoli-localized protein. The tissue expression profile analysis showed that Fkbp19 was expressed in several exocrine glands and the subcellular localization of Fkbp19 protein was found to be largely in the endoplasmic reticulum and nucleoli. All these results showed that Fkbp19 expression was closely correlated with the synthesis and secretion of the protein in cell.In order to elucidate the possible role of Fkbp19 in tumor genesis, the RNAi technology was employed to knockdown the expression of the Fkbp19 in cell line (ZR75-1) in which Fkbp19 was found to be highest expressed by RT-PCR. The expression of Fkbp19 was successfully suppressed in ZR75-1 cells stably transfected with shRNA constructs as consistently shown by two different methods. MTT and FCM were used to detect the ability of proliferation of the Fkbp-knockdown cells, and the result showed that proliferation of those cells was decreased and cell cycle was arrested at G1 phase. Western blot showed the expression of CDK4 and CCND1, two crucial factors that help cells go through the G1/S check point, was decreased. Transwell experiment was use to analyze the ability of the migration of the cells, the result showed that the ability of the migration decreased dramatically upon Fkbp19 knockdown.Considering commercial antibody for Fkbp19 is not available yet, we prepare the antibody by ourselves. After prokaryotic expression induced by the IPTG, protein purification with the His-tag purification system, immunizing the rabbits and gathering the serum, we obtained the anti-serum against Fkbp19, which could be used to detect endogenous protein of Fkbp19 by Western blot. In sum, Fkbp19 was highly expressed in the HCC tissue of mouse and human, and its expression in normal tissues is correlate...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221985
Collection生命科学学院
Recommended Citation
GB/T 7714
范开吉. Fkbp19基因的初步研究及抗体制备[D]. 兰州. 兰州大学,2010.
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