本文關(guān)鍵詞：異檸檬酸裂解酶基因克隆與表達特征研究　出處：《長春工業(yè)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 結(jié)核分枝桿菌 異檸檬酸裂解酶 乙醛酸旁路 原核表達

【摘要】：近年來世界范圍內(nèi)結(jié)核病發(fā)病率明顯上升,結(jié)核病感染的持留狀態(tài)這一問題普遍存在。異檸檬酸裂解酶,一種由ICL基因編碼,作為乙醛酸代謝途徑中的關(guān)鍵酶,在結(jié)核分枝桿菌感染的巨噬細(xì)胞及其在人體內(nèi)持續(xù)感染起關(guān)鍵支持作用。結(jié)核分枝桿菌代謝途徑的這一關(guān)鍵酶已經(jīng)被作為抗結(jié)核藥物研究和開發(fā)的新型靶點,這就需要表達出具有生物學(xué)活性的重組異檸檬酸裂解酶。 為表達出具有生物活性的重組異檸檬酸裂解酶,本研究以結(jié)核分枝桿菌H37Rv基因組為模板,通過PCR反應(yīng)擴增該菌株的ICL基因,并將測序正確的ICL基因,克隆入原核表達載體pET-28a(+)中,取名為pET28-ic1。重組異檸檬酸裂解酶在大腸桿菌BL21(DE3)中表達出來。通過Ni-NTA親和層析柱純化,以獲得純化的結(jié)核分枝桿菌H37Rv異檸檬酸裂解酶重組蛋白。對重組蛋白ICL進行酶學(xué)性質(zhì)分析,證明該重組蛋白其具有異檸檬酸裂解酶活性。 現(xiàn)已成功在結(jié)核分枝桿菌H37Rv中將異檸檬酸裂解酶表達出來,并鑒定得出純化的重組異檸檬酸裂解酶具有生物活性。為了測定異檸檬酸裂解酶的活性,需要實時測定NADH量的減少。異檸檬酸在異檸檬酸裂解酶催化下形成乙醛酸,乙醛酸可通過乳酸脫氫酶和NADH還原成甘醇酸酯。NADH的減少則通過加入甲佨染料MTS(MTS／PMS比率為100：1),并在490nm處測定吸光度值來確定。純化的異檸檬酸裂解酶重組蛋白酶活為1.84×102μmol×mg-1×min-1。異檸檬酸裂解酶重組蛋白生物活性的最適PH值為7.4。 我們應(yīng)用原核表達系統(tǒng),在結(jié)核分枝桿菌H37Rv中將ICL克隆和表達出來,純化出了具有生物學(xué)活性的結(jié)核分枝桿菌H37Rv可溶性異檸檬酸裂解酶,并對其酶學(xué)性質(zhì)進行了研究,酶學(xué)性質(zhì)鑒定獲得了具有生物學(xué)活性的重組蛋白。本研究為該酶免疫學(xué)研究及新型抗結(jié)核藥物的開發(fā)與設(shè)計提供設(shè)計方案與理論基礎(chǔ)。
[Abstract]:In recent years, the incidence of tuberculosis has increased worldwide, and the persistence of TB infection is widespread. Isocitrate lyase, a gene encoded by ICL. As a key enzyme in glyoxylic acid metabolism pathway. Macrophages infected by Mycobacterium tuberculosis and their persistent infection in humans play a key supporting role. This key enzyme of Mycobacterium tuberculosis metabolic pathway has been used as a new target for the research and development of anti-tuberculosis drugs. This requires the expression of recombinant isocitrate lyase with biological activity. In order to express recombinant isocitrate lyase with biological activity, the ICL gene of Mycobacterium tuberculosis H37Rv was amplified by PCR reaction. The correct ICL gene was cloned into the prokaryotic expression vector pET-28a (). The recombinant isocitrate lyase was expressed in E. coli BL21DE3 and purified by Ni-NTA affinity chromatography. The purified recombinant protein of Mycobacterium tuberculosis H37Rv isocitrate lyase was obtained. The enzymatic properties of the recombinant protein ICL showed that the recombinant protein had isocitrate lyase activity. The isocitrate lyase has been successfully expressed in Mycobacterium tuberculosis H37Rv, and the purified recombinant isocitrate lyase has been identified to have biological activity in order to determine the activity of isocitrate lyase. The amount of NADH needed to be determined in real time. Isocitric acid was catalyzed by isocitrate lyase to form glyoxylic acid. Glyoxylic acid can be reduced by lactate dehydrogenase and NADH to glycolate. Nadh is reduced by adding MTS(MTS/PMS ratio of 100: 1). The purified recombinant protease activity of isocitrate lyase was 1.84 脳 102 渭 mol 脳 mg-1 脳 min-1. The recombinant protein of isocitrate lyase was determined by measuring the absorbance at 490 nm. The optimum PH value of biological activity is 7.4. ICL was cloned and expressed in Mycobacterium tuberculosis H37Rv using prokaryotic expression system. The soluble isocitrate lyase of Mycobacterium tuberculosis H37Rv with biological activity was purified and its enzymatic properties were studied. The recombinant protein with biological activity was obtained by the identification of enzymatic properties. This study provides a design scheme and theoretical basis for the immunological study of the enzyme and the development and design of novel antituberculous drugs.
【學(xué)位授予單位】：長春工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

【引證文獻】

相關(guān)碩士學(xué)位論文 前2條

1 牛雪;以異檸檬酸裂解酶為靶點篩選肽類抑制劑[D];長春工業(yè)大學(xué);2011年

2 趙韞慧;異檸檬酸裂解酶肽類抑制劑的優(yōu)化篩選[D];長春工業(yè)大學(xué);2012年

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本文編號：1372044