|Alternative Title||The primary research of Fkbp19 and the preparation of antibody
|Place of Conferral||兰州
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。
|Other Abstract||Through 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...|
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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