【作者】 康慧芳；

【导师】 牛勃；

【作者基本信息】 山西医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的1.双胸蚓组织中核酸酶粗提物的制备及核酸酶的纯化2.核酸酶粗提物琼脂糖凝胶测活体系的建立3.纯化核酸酶酶学性质的研究4.双胸蚓组织核酸酶底物特异性的初步研究方法1.双胸蚓组织中核酸酶粗提物的制备及核酸酶的纯化以SDS-PAGE-DNA功能胶和琼脂糖凝胶电泳测活相结合作为双胸蚓组织核酸酶提取过程中核酸酶活性的监测体系。以碳酸铵处理,热变性,酸变性及丙酮沉淀后所得粗提物为起点,对核酸酶依次进行柱层析、电泳纯化制备双胸蚓核酸酶样蛋白质纯品。2.核酸酶粗提物琼脂糖凝胶测活体系的建立以琼脂糖凝胶电泳作为主要测活体系,研究核酸酶粗提物的最适反应条件。3.纯化核酸酶酶学性质的研究采用SDS-PAGE电泳法测定酶分子量,毛细管等点聚焦电泳法测定酶等电点,酶活性测定等方法测定核酸酶的最适pH、最适温度、温度稳定性、激动剂、抑制剂,计算酶的动力学常数。4.双胸蚓组织核酸酶底物特异性的研究测定DNase E在最适反应条件下对不同底物的降解速率,证明DNase E降解底物的选择性;应用琼脂糖凝胶电泳对DNase E水解双链环状DNA后的产物进行鉴定,阐明DNase E的水解方式。结果1.双胸蚓组织中核酸酶粗提物的制备及核酸酶的纯化应用本室建立的SDS-PAGE-DNA功能胶测活方法对核酸酶提取过程进行监测。在提取过程中产生两组核酸酶,在SDS-PAGE-DNA功能胶中迁移率较慢的一组采用碳酸铵处理+酸变性+热变性+丙酮沉淀的提取方法来获得较好的层析样品。样品依次通过四步柱层析,PAGE-DNA功能胶纯化得到电泳纯的双胸蚓核酸酶样蛋白质纯品,命名为DNase E（Earthworm DNase）。2.核酸酶粗提物琼脂糖凝胶测活体系的建立以琼脂糖凝胶电泳法作为主要测活手段,确定了2μl质粒+10μl样品,样品溶于0.1 M pH 5.2NaAc（含20mM Mg2+）缓冲液,37℃共孵育30min后行琼脂糖凝胶电泳的测活体系。3.纯化核酸酶酶学性质的研究对DNase E的酶学性质进行了初步研究,结果表明其分子量约为32kDa,等电点为6.05左右,以小牛胸腺DNA为底物时,反应的最适pH值为5.2,最适温度为42℃,其Km值为0.152mg/ml。20mM Mg²⁺,Ca²⁺,Mn²⁺对其活性有一定的激活作用,Zn²⁺,咪唑,EDTA,K+,Cu²⁺对其活性有一定的抑制作用,SDS-PAGE的结果表明DNase E为寡聚肽蛋白质,它对酸、碱、有机溶剂稳定。4.双胸蚓组织核酸酶底物特异性的研究通过DNase E在最适反应条件下对几种不同底物降解速率的研究表明DNase E降解底物存在差异性。应用琼脂糖凝胶电泳对DNase E水解双链环状DNA后的产物进行鉴定,证明了DNase E属于核酸内切酶。结论1.确立了双胸蚓组织核酸酶粗提液的制备工艺,通过柱层析及电泳纯化的方法,获得了一种核酸酶,命名为DNase E（电泳纯）,分子量约为32kDa,等电点为6.05左右,反应的最适pH值为5.2,最适温度为42℃,20mM Mg²⁺,Ca²⁺,Mn²⁺对其活性有一定的激活作用,Zn²⁺,咪唑,EDTA,K+,Cu²⁺对其活性有一定的抑制作用。2.确立了核酸酶粗提物的琼脂糖凝胶电泳测活体系。实验结果表明,DNase E无序列特异性,属非特异核酸酶类。初步得出DNase E在底物水解方面的差异性,并证明了DNase E属于核酸内切酶。更多还原

【Abstract】 Objectives1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification.2. To establish the agarose gel electrophoresis system.3. Study on enzymology qualities of the nuclease.4. A preliminary study on substrate specificity of Lumbricus Bimastus nuclease.Methods1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification Using DNA-casting SDS-PAGE and agarose gel to monitor the activity of the nuclease in the process of extraction.After ammonium carbonate processing, thermal denaturation, acid denaturation and acetone precipitation,the rude extracts of nucleases were purified by Column chromatography and electrophoresis.2. To establish the agarose gel electrophoresis systemUsing agarose gel electrophoresis system to monitor the activity of the nuclease, to study its optimum reaction conditions.3. Study on enzymology qualities of the nucleaseUsing SDS-PAGE gel electrophoresis to measure molecular weight, CE-IEF to measure pI, assay of enzyme activity to determine optimum pH, optimum temperature, temperature stability, activator, inhibitor, and to calculate its Km values.4. A preliminary study on substrate specificity of Lumbricus Bimastus nucleaseTo measure degrade rate of DNase E to several different substrate in optimal reaction condition to demonstrate the substrate selectivity of DNase E. Agarose gel electrophoresis was used to investigate the products of DNase E processing double strands cyclic DNA to investigate the action of DNase E.Results1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification Two groups of nucleases with different characters lied in the DNA-casting SDS-PAGE. The group of low migration nucleases were prepared by ammonium carbonate processing +acid denaturation + thermal denaturation + acetone extraction. Through four steps of column chromatography and electrophoresis purification, one kind of nuclease（named DNase E） was obtained. 2. To establish the agarose gel electrophoresis systemTo identify the agarose gel electrophoresis system that using plasmid 2μl +10μl as samples(sample dissolved in 0.1 M pH 5.2NaAc buffer (containing 20 mM Mg²⁺)), then incubated for 30 min in 37℃.3. Study on enzymology qualities of the nucleaseThe enzymatic study results of DNase E showed that the molecular weight of DNase E was approximately 32 kDa, its isoelectric point was about 6.05. When the substrate was the calf thymus DNA, its optimum temperature was 42℃, optimum pH was 5.2, and its Km values was 0.152mg/ml. 20mM Mg²⁺、Ca²⁺、Mn²⁺ could activate its activity respectively. Zn²⁺, iminazole, EDTA, K+, Cu²⁺ could inhibit its activity respectively. The SDS-PAGE showed that DNase E was a oligomeric protein, it was stable to acid, alkali, and organic solvents.4. A preliminary study on substrate specificity of Lumbricus Bimastus nuclease DNase E degradation studies showed that there were differences in degradation rate of the DNase E to different substrates. The results indicated that DNase E belonging to endonuclease.Conclusion1. Established a preparation technics of rude extracts of Lumbricus Bimastus nucleases. One kind of nuclease was obtained by a series of purification methods, named DNase E （electrophoresis pure）.2. The molecular weight of DNase E was approximately 32kDa, its isoelectric point was about 6.05. When the substrate was the calf thymus DNA, its optimum temperature was 42℃, optimum pH was 5.2. 20mM Mg²⁺、Ca²⁺、Mn²⁺ could activate its activity respectively. Zn²⁺, iminazole, EDTA, K+, Cu²⁺could inhibit its activity respectively. The experimental results showed that DNase E had no sequence specificity, it was a non-specific nuclease. There was difference in substrate hydrolysis. DNase E belonged to endonucleases.更多还原