本文選題：Caspase-3的原核表達 + 蛋白酶抑制劑�。� 參考：《吉林大學》2010年碩士論文

【摘要】： Caspase家族是一類半胱氨酸-天冬氨酸蛋白酶,主要存在于細胞質(zhì)中,能特異性斷裂蛋白的天冬氨酸位點,是細胞凋亡中的關鍵因子。尤其caspase-3作為凋亡的執(zhí)行者,能夠啟動凋亡的信號并級聯(lián)放大,從而引起細胞凋亡。細胞凋亡不僅僅是多細胞生物體的正常機能,而且還能夠引發(fā)各種疾病:通過神經(jīng)元細胞的凋亡可引起神經(jīng)退行性疾病,如阿爾茨海默癥、帕金森癥等;由于心肌細胞的凋亡可引發(fā)一系列心血管疾病,常見的有動脈粥樣硬化、心肌炎、心律不齊等等;還有報道細胞凋亡與Ι型糖尿病也相關。因此,開發(fā)以caspase-3為靶點的抑制劑對于治療這些重大疾病具有廣闊的前景。 本論文首先從caspase-3重組質(zhì)粒的構建開始,將大小亞基作為一整體進行低溫誘導、促可溶的表達,嘗試了幾種載體和不同宿主內(nèi)的表達,最終發(fā)現(xiàn)以pET-28b(+)為載體,在BLP宿主中可以相對的提高可溶形式的目的蛋白表達量。重組蛋白經(jīng)金屬鏊和層析分離純化,利用酶標儀檢測活性。并對重組caspase-3蛋白酶進行了酶學表征。然后以caspase-3為分子靶點,以我國豐富的中藥資源為基礎,利用已建立的中藥文庫,應用生物化學、酶學方法對中藥文庫進行初步的篩選和鑒定,以期篩選出具有理想效果的天然產(chǎn)物抑制劑,從而開發(fā)出以caspase-3為靶點的新藥。實驗結果表明,我們獲得了高純度和高活性的重組蛋白caspase-3,并通過酶學方法得到表征,利用酶學手段,我們從部分中藥中初步篩選出了對caspase-3有明顯抑制作用的中藥:蓮須和雞血藤。這些結果表明,我們成功表達的重組caspase-3蛋白,能夠應用于中藥文庫caspase-3抑制劑的篩選,這為中藥理論的研究提供了實驗依據(jù),對藥物的開發(fā)和創(chuàng)新具有一定積極的影響。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

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