Caspase-3原核表达及其抑制剂的筛选

发布时间：2018-05-08 23:26

本文选题：Caspase-3的原核表达 + 蛋白酶抑制剂　；参考：《吉林大学》2010年硕士论文

【摘要】： Caspase家族是一类半胱氨酸-天冬氨酸蛋白酶,主要存在于细胞质中,能特异性断裂蛋白的天冬氨酸位点,是细胞凋亡中的关键因子。尤其caspase-3作为凋亡的执行者,能够启动凋亡的信号并级联放大,从而引起细胞凋亡。细胞凋亡不仅仅是多细胞生物体的正常机能,而且还能够引发各种疾病:通过神经元细胞的凋亡可引起神经退行性疾病,如阿尔茨海默症、帕金森症等;由于心肌细胞的凋亡可引发一系列心血管疾病,常见的有动脉粥样硬化、心肌炎、心律不齐等等;还有报道细胞凋亡与Ι型糖尿病也相关。因此,开发以caspase-3为靶点的抑制剂对于治疗这些重大疾病具有广阔的前景。本论文首先从caspase-3重组质粒的构建开始,将大小亚基作为一整体进行低温诱导、促可溶的表达,尝试了几种载体和不同宿主内的表达,最终发现以pET-28b(+)为载体,在BLP宿主中可以相对的提高可溶形式的目的蛋白表达量。重组蛋白经金属鏊和层析分离纯化,利用酶标仪检测活性。并对重组caspase-3蛋白酶进行了酶学表征。然后以caspase-3为分子靶点,以我国丰富的中药资源为基础,利用已建立的中药文库,应用生物化学、酶学方法对中药文库进行初步的筛选和鉴定,以期筛选出具有理想效果的天然产物抑制剂,从而开发出以caspase-3为靶点的新药。实验结果表明,我们获得了高纯度和高活性的重组蛋白caspase-3,并通过酶学方法得到表征,利用酶学手段,我们从部分中药中初步筛选出了对caspase-3有明显抑制作用的中药:莲须和鸡血藤。这些结果表明,我们成功表达的重组caspase-3蛋白,能够应用于中药文库caspase-3抑制剂的筛选,这为中药理论的研究提供了实验依据,对药物的开发和创新具有一定积极的影响。
[Abstract]:Caspase family is a kind of cysteine-aspartic protease, which mainly exists in the cytoplasm. The aspartic acid site which can specifically break the protein is the key factor in cell apoptosis. Especially, as the executor of apoptosis, caspase-3 can initiate the signal of apoptosis and cascade amplification, thus causing apoptosis. Apoptosis is not only the normal function of multicellular organisms, but also can cause various diseases: neuronal apoptosis can cause neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and so on; Cardiomyocyte apoptosis can lead to a series of cardiovascular diseases, such as atherosclerosis, myocarditis, arrhythmia, etc. Apoptosis is also reported to be associated with type I diabetes. Therefore, the development of caspase-3-targeted inhibitors has broad prospects for the treatment of these major diseases. Starting with the construction of caspase-3 recombinant plasmid, the small and small subunits were induced as a whole to promote soluble expression. Several vectors and different host vectors were used to express pET-28b (), and pET-28b () was used as a vector. The expression of soluble target protein in BLP host can be increased relatively. The recombinant protein was purified by metal chelate and chromatography. The recombinant caspase-3 protease was characterized by enzyme. Then the caspase-3 was used as the molecular target, based on the rich Chinese medicine resources in China, and the established Chinese medicine library was used to screen and identify the Chinese medicine library by biochemical and enzymatic methods. In order to screen natural product inhibitors with ideal effect, a new drug targeting caspase-3 was developed. The results showed that the recombinant protein caspase-3 with high purity and activity was obtained and characterized by enzymatic method. Some Chinese medicines with obvious inhibitory effect on caspase-3 were selected from some Chinese medicines: Liansuke and Hicken-blood Rattan. These results indicate that the recombinant caspase-3 protein we successfully expressed can be applied to the screening of caspase-3 inhibitors in Chinese medicine library, which provides experimental basis for the theoretical study of Chinese medicine and has a certain positive effect on the development and innovation of drugs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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