本文選題：炎癥Caspase　切入點：Caspase-1　出處：《華東師范大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：炎癥Caspase(Cystine proteases with aspartic acid substrate specificity)是Caspases家族中一類與炎癥反應(yīng)重要相關(guān)的蛋白酶，其主要功能是產(chǎn)生成熟的促炎癥細胞因子IL-1β和IL-18。成熟的IL-1β和IL-18具有廣泛的生物活性，并與多種疾病有關(guān)，如各種病毒、細菌、真菌等微生物的感染、外傷、心肌梗塞、肝炎、胰腺炎、關(guān)節(jié)炎、神經(jīng)炎癥和神經(jīng)退行性疾病。炎癥Caspase包括Caspase-1、-4、-5、-11、-12。其中最具代表性的是Caspase-1，是切割I(lǐng)L-1β和IL-18的高效特異的蛋白酶。體外和體內(nèi)實驗結(jié)果表明抑制炎癥Caspase，尤其是Caspase-1的活性，能緩解炎癥過度引起的各種疾病，如關(guān)節(jié)炎，牛皮癬、神經(jīng)損傷等。因此，Caspase-1成為治療炎癥過度性疾病的重要靶點，尋找Caspase-1高效高選擇性的抑制劑為治療這類疾病提供了新的途徑。本論文的目的在于表達和純化Caspase-1重組蛋白，研究其酶學(xué)性質(zhì)，并在此基礎(chǔ)上建立Caspase-1抑制劑分子水平的高通量篩選模型，并通過對國家化合物樣品庫的篩選，，尋找高親和力的Caspase-1抑制劑，通過對其性質(zhì)進行研究，為進一步的化合物結(jié)構(gòu)改造以及細胞和動物水平的藥理學(xué)、藥效學(xué)研究打下基礎(chǔ)。 本文的研究內(nèi)容包括用RT-PCR方法克隆Caspase-1全長基因，并根據(jù)文獻報道將Caspase-1的第381位氨基酸突變使重組蛋白更穩(wěn)定。將Caspase-1的催化區(qū)域克隆到表達載體pET21b上，并在重組蛋白的N端加上(His)_6標簽。將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21-CodonPlus(DE3)后經(jīng)IPTG誘導(dǎo)表達。通過Ni~(2+)親和層析柱純化，獲得較純凈的活性形式Caspase-1重組蛋白。通過對Caspase-1的一系列酶學(xué)特性，包括DTT的影響、pH依賴性、動力學(xué)分析及陽性抑制劑抑制作用分析，優(yōu)化Caspase-1的活性檢測方法，建立了Caspase-1抑制劑高通量篩選的標準化操作流程。通過對國家化合物樣品庫中的47360個樣品進行篩選，得到了16個IC_(50)低于10μM抑制活性較好的化合物。其中一活性化合物MXQ-1的IC_(50)為163nM，為新型的Caspase-1抑制劑。通過分子水平酶動力學(xué)方法對其進行抑制性質(zhì)研究，發(fā)現(xiàn)該化合物為可逆的、慢結(jié)合型廣譜Caspase抑制劑，其與Caspase-1之間的相互作用模式為簡單型相互作用，并不發(fā)生酶的異構(gòu)化過程。這一新型Caspase-1抑制劑的發(fā)現(xiàn)和相關(guān)抑制性質(zhì)的研究為其進一步結(jié)構(gòu)優(yōu)化和藥理學(xué)、藥效學(xué)研究打下堅實的基礎(chǔ)。
[Abstract]:Inflammatory Caspase(Cystine proteases with aspartic acid substrate specificity (Caspase(Cystine proteases with aspartic acid substrate specificity) is a kind of protease related to inflammation in Caspases family. Its main function is to produce mature inflammatory cytokines IL-1 尾 and IL-18.The mature IL-1 尾 and IL-18 have a wide range of biological activities and are related to many diseases. Such as viruses, bacteria, fungi and other microbial infections, trauma, myocardial infarction, hepatitis, pancreatitis, arthritis, Inflammation and neurodegenerative diseases. Inflammatory Caspase includes Caspase-1, caspase-1, caspase-1, caspase-1, a highly efficient and specific protease that cleans IL-1 尾 and IL-18. In vitro and in vivo experiments have shown that it inhibits the activity of inflammatory caspase, especially Caspase-1. It can relieve various diseases caused by excessive inflammation, such as arthritis, psoriasis, nerve damage, etc. Therefore, Caspase-1 has become an important target for the treatment of excessive inflammatory diseases. The aim of this paper is to express and purify the recombinant Caspase-1 protein and study its enzymatic properties. On the basis of this, a high throughput screening model of Caspase-1 inhibitors at molecular level was established, and a high affinity Caspase-1 inhibitor was found by screening the national compound sample bank, and its properties were studied. It provides a basis for further structural modification of compounds and pharmacological and pharmacodynamic studies at cellular and animal levels. In this paper, the full-length Caspase-1 gene was cloned by RT-PCR, and the 381st amino acid mutation of Caspase-1 was reported to make the recombinant protein more stable. The catalytic region of Caspase-1 was cloned into the expression vector pET21b. The recombinant plasmid was transformed into Escherichia coli BL21-CodonPlusDE3 and expressed by IPTG. The recombinant protein was purified by Ni~(2 affinity chromatography. A series of enzymatic properties of Caspase-1 were obtained. Including the effect of DTT on pH dependence, kinetic analysis and inhibition analysis of positive inhibitors, optimization of Caspase-1 activity detection method, A standardized procedure for high throughput screening of Caspase-1 inhibitors was established. 47360 samples from the national sample bank were screened. 16 compounds with better inhibitory activity than 10 渭 M were obtained, in which one active compound, MXQ-1, was 163nM, which was a new type of Caspase-1 inhibitor. The inhibitory properties of the compound were studied by enzyme kinetic method at molecular level, and it was found that the compound was reversible. A slow binding broad-spectrum Caspase inhibitor whose interaction mode with Caspase-1 is simple. There is no isomerization process of the enzyme. The discovery of this new Caspase-1 inhibitor and the study of its related inhibitory properties lay a solid foundation for its further structural optimization pharmacological and pharmacodynamic studies.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 蔡龍飛;基于流動注射梯度技術(shù)的液滴微流控系統(tǒng)及其在酶抑制劑篩選和酶反應(yīng)動力學(xué)研究中的應(yīng)用[D];浙江大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 王燕燕;Caspase-3原核表達及其抑制劑的篩選[D];吉林大學(xué);2010年

本文編號：1613433