一种新型的脱氧核糖核酸酶的分离纯化、性质及免疫功能的研究

【作者】 刘志贞；

【导师】 牛勃； 程牛亮；

【作者基本信息】 山西医科大学 ， 生理学， 2009， 博士

【摘要】 蚯蚓核酸酶系本课题组近年来在研究蚯蚓过程中发现的一组具有抗病毒活性的核酸酶类。初步研究结果提示,该类酶极具成为新型的、独特的、具有高效抗病毒作用的杀灭微生物的中药生物制剂的良好前景,并有助于人类在发现和探索新型抗微生物制剂和研究抗病毒制剂的征途上另辟蹊径。近年来我们已从该组分纯化出三种脱氧核糖核酸酶,并对三种脱氧核糖核酸酶进行了理化性质、生化表征特征的研究及基因鉴定。肽质量指纹图谱结果为新蛋白。ELMWD（Earthworm Low molecular weight deoxyribonuclease）是本研究在优化脱氧核糖核酸酶制备工艺的基础上通过功能胶新发现的一种分子量相对较小的脱氧核糖核酸酶。文献检索表明,地龙中的核酸酶是本课题组率先发现并展开研究的,ELMWD是其中的一种。本课题将进一步从纯化工艺、酶学性质、蛋白表征特征、免疫功能等生物化学、分子生物学和分子药理学的内容进行研究。ELMWD的发现和机制研究,尚未见其它文献报道。研究方法及结果一、蚯蚓组织脱氧核糖核酸酶测活、提取工艺的优化及一种小分子量脱氧核糖核酸酶（ELMWD）的纯化（一）蚯蚓脱氧核糖核酸酶测活方法的扩充及改进测活是核酸酶纯化工作中最重要的部分,本研究提出了采用兼有分离及监测酶种类的SDS-PAGE功能胶测活方法,以DNaseI作为标准品,摸索出了最佳的实验条件及去除SDS对酶活性的影响方法,为后续的研究奠定了方法基础。（二）蚯蚓脱氧核糖核酸酶层析样品制备工艺的优化层析样品的制备是分离纯化的前提,课题组前期实验制备的脱氧核糖核酸酶层析样品具有杂蛋白、粘蛋白及色素较多的缺点,影响了后续层析分离过程,针对上述三点本研究提出了三条工艺路线并进行验证。与前期层析样品制备的主要区别在于探索了碳酸铵抽提、热变性、丙酮分级沉淀、纯丙酮洗涤、硫酸铵分级沉淀及酸变性等处理因素。采用SDS-PAGE功能胶和琼脂糖凝胶电泳法监测酶的种类及活性的变化情况;以脱氧核糖核酸酶活性与种类的保留、粘性物质、杂蛋白的去除为主要目标,比较不同处理方法的结果。结果在SDS-PAGE功能胶监测的过程中发现了两组电泳迁移率不同的脱氧核糖核酸酶,这两组脱氧核糖核酸酶对不同的处理因素耐受情况不同,针对这两组迁移率不同的脱氧核糖核酸酶分别提出了两套不同的分离纯化路线。大分子量组采用机械匀浆、碳酸铵抽提、60℃,10min热变性、pH3.8丙酮分级沉淀（或者pH4.6丙酮50%一次性沉淀）的工艺路线;小分子量组采用机械匀浆、碳酸铵抽提、硫酸铵分级沉淀、丙酮沉淀的工艺路线。（三）蚯蚓组织脱氧核糖核酸酶（ELMWD）的层析及电泳纯化利用前述方法得到小分子量脱氧核糖核酸酶层析样品,进一步利用DEAE阴离子交换层析、疏水层析的方法分离脱氧核糖核酸酶,以功能胶及琼脂糖凝胶电泳测活的方法监测活性峰的收集。在层析纯化的基础上,采用双向电泳及双向功能胶等技术相结合的方法,从蚯蚓组织中纯化出了一种DNA酶,命名为ELMWD。二、一种小分子量蚯蚓脱氧核糖核酸酶（ELMWD）的生化表征特征及酶促动力学研究（一） ELMWD酶促反应动力学研究利用紫外分光光度法、琼脂糖凝胶电泳法对ELMWD进行最适pH及pH稳定性、最适温度及温度稳定性的研究,研究了不同底物浓度、不同的金属离子对酶促反应速度的影响并给出了各影响因素的参数。结果ELMWD最适pH为5.2,在pH4.0-7.0的范围内较稳定;最适温度为37℃,且对温度比较敏感,在40℃范围内活性相对稳定;Mg²⁺、Ca²⁺、Mn²⁺、EDTA对酶的活性有不同程度的抑制作用,5mM的K⁺对酶的活性有激活作用;当以小牛胸腺DNA为底物时酶的Km=0.040μg/μl,Vmax=0.059μg/μl·min-1,当以质粒DNA为底物时,酶的Km=0.090μg/μl,Vmax=0.11μg/μl·min-1。并且对RNA没有降解作用。这些参数与已发现的各种DNases相比较有明显差别。（二） ELMWD生化表征性质的研究采用SDS-PAGE电泳、生物质谱方法对ELMWD的蛋白性质包括纯度、分子量、肽质量指纹图谱进行测定;利用双向电泳及毛细管电泳法测定ELMWD的等电点;得出ELMWD的纯度在99.5%以上;等电点为4.5;分子量大小为20759.417;肽质量指纹图谱搜索结果未见有意义的高分匹配蛋白。三、蚯蚓脱氧核糖核酸酶与固有免疫系统关系的研究本部分围绕蚯蚓脱氧核糖核酸酶与固有免疫的关系进行了以下几个方面的研究:研究了脱氧核糖核酸酶的蚯蚓组织分布,为进一步理解其功能提供依据;从与固有免疫相衔接的抵抗外来物质（主要是病毒和细菌）,清除体内衰老和畸变细胞（肿瘤细胞）两个角度研究了蚯蚓脱氧核糖核酸酶在蚯蚓固有免疫中的作用和地位。（一） ELMWD及蚯蚓脱氧核糖核酸酶的组织分布采用免疫组化、免疫印迹、单相酶扩散、功能胶活性测定法研究了ELMWD及总脱氧核糖核酸酶在蚯蚓组织中的分布。结果证明ELMWD在赤子爱胜蚓组织内的分布是有部位特异性的。免疫组化结果表明ELMWD主要分布于赤子爱胜蚓的皮肤层及脏层粘膜,构成了内外两层屏障,免疫印迹及活性测定结果表明ELMWD及总脱氧核糖核酸酶在蚯蚓组织分布的顺序如下:消化管道,体腔液,体腔壁,体细胞,循环系统,生殖系统,神经系统。这样的分布特点与其功能有着密切的联系,提示蚯蚓脱氧核糖核酸酶主要参与了蚯蚓的免疫及消化外界微生物的功能。（二）蚯蚓体腔液活性提取物及ELMWD杀菌活性及机制的初步探讨体外杀菌实验采用细菌诱导前后蚯蚓体腔液活性提取物对细菌（G-和G+菌、耐药菌）结构逐层降解的方法。荧光分光光度法、分光光度法分别检测对革兰氏阴性菌外膜,细胞壁肽聚糖成分和细胞膜成分的降解作用;琼脂糖凝胶电泳法检测对细菌基因组及质粒的降解作用;采用免疫印迹方法检测细菌诱导前后ELMWD表达情况;进一步利用生物膜形成实验检测了蚯蚓DNase ELMWD抑菌机制的研究。结果证明ELMWD细菌诱导后表达增高;蚯蚓体腔液活性提取物能够由外向内逐层降解细菌的结构而起到杀灭细菌的作用;ELMWD独立没有杀菌作用,但是能够协同其他免疫分子的杀菌活性,并且能够协同溶菌酶降解细菌的基因组;可以抑制细菌生物膜的形成。（三）蚯蚓体腔液活性提取物及ELMWD抗肿瘤活性及机制的初步探讨本章内容利用MTT和SRB方法检测了蚯蚓体腔液活性提取物及ELMWD抑制四种肿瘤细胞增殖的活性;采用Hochest荧光染色和单细胞凝胶电泳检测肿瘤细胞基因组的情况;采用Transwell Migration实验检测肿瘤的转移情况,并且还利用动物实验检测ELMWD抗肿瘤结果。结果证明蚯蚓体腔液活性提取物有显著的抑制肿瘤细胞增殖的活性;肿瘤细胞基因组被降解为小的碎片。流式细胞仪技术检测到凋亡细胞存在,认为对肿瘤细胞增殖的抑制作用主要通过将细胞阻滞在S期以及通过细胞凋亡所致。而ELMWD体外对抑制肿瘤细胞的增殖作用不显著,但是体内可以显著抑制肿瘤的生长作用。体外还可以显著协同活性提取物的抗肿瘤作用,而且对肿瘤的侵袭转移具有明显的抑制作用。（四）蚯蚓体腔液粗提物及ELMWD杀抗病毒活性及机制的初步探讨本章内容利用病毒噬斑实验及阴阳离子交换层析的方法从蚯蚓组织分离出三组具有非特异性抗病毒（流感病毒、腺病毒、轮状病毒）活性的成分,这三组成分分别具有高活性的蛋白酶、溶菌酶和核酸酶（DNase和RNase）。三组成分混合以后抗病毒能力并不能恢复到蚯蚓体腔液活性提取物的水平,但是要高于每个活性组。本研究同时测定了蚯蚓DNaseELMWD的抗病毒能力,证明ELMWD抗肿瘤活性不明显,但是可以显著协同活性提取物的抗病毒能力。其中核酸酶成分组及ELMWD具有降解病毒遗传物质的能力。结论1.在前期的实验基础上,扩充了一种酶活性测定方法,提出并验证了一种分辨率高的、能够监测酶种类变化的兼有测活作用的SDS-PAGE-DNA功能胶方法,为后续的实验奠定了方法基础。2.优化并建立了蚯蚓组织中两组迁移率不同的脱氧核糖核酸酶层析样品的制备工艺。3.建立了一套成熟的小分子量的蚯蚓脱氧核糖核酸酶（ELMWD）的纯化工艺。4.获得了一种新型的纯度较高的脱氧核糖核酸酶（ELMWD）,分子量为20759Da,等电点4.5,最适pH为5.2,最适温度为40℃,对线状小牛胸腺DNA比对环状质粒DNA的亲和力高,对RNA没有降解作用。酶学性质及质谱鉴定结果提示这种酶是一种不同于其他的已发现的DNase的脱氧核糖核酸酶。5.蚯蚓脱氧核糖核酸酶属于蚯蚓固有免疫的一个很重要的元素,脱氧核糖核酸酶与其他免疫分子协同或独立的在降解内外源废弃细胞及微生物的遗传物质上发挥了不可替代的作用。研究开发蚯蚓体内的脱氧核糖核酸酶具有很重要的意义。更多还原

【Abstract】 Earthworm nucleases are found during the process of extract anti-antiviral substances from earthworm.From what we have studied we can conclude that these group of nucleases have promising future to be anti-microbial and anti-tumor traditional medicines.These will help people to get a new idea in the probing new medicine of anti-virus.We have purified three kinds of DNases,PMF results have showed they are novel proteins.Earthworm low molecular weight deoxyribonuclease（ELMWD） is a kind of novel DNase that we have found.From the best of we have known,ELMWD is the first time to be studied.Our study will focus on the purify technique,Enzymatic mechanism,immune function of ELMWD.The methods and the results are briefly stated as followings:Methods and Results:1.Study on optimization deoxyribonuclease purify technology and purification Earthworm low molecular weight deoxyribonuclease（ELMWD）1.1 The study on expansion a kind of DNase activity measurementIn this chaptor,we introduced a new-type of activity measurement which have high resolution and can monitor the change of deoxyribonucleic number.DNaseI has been used to test the process.We have studied on how to eliminate the SDS impact on the activity of DNase.1.2 The optimization of deoxyribonuclease Chromatography sample preparation technologyThe nuclease Chromatography sample prepared in prophase experiment had much hybridprotein,mucoprotein,pigment which can affect the following chromatographic fractionation process,especially lead to the irreversible destroy on the column material.According to those information,three technical routes were proposed and verificated. Function gel and agar gel electrophoresis were used to monitor the numbers and activities change;Different methods were compared taking the activity to be reserved,the stickum and hybridprotein were removed as the main objective.The result showed that there were two kinds of DNases according to the electrophoresis mobility which had different tolerance to different elements.Two kinds of purify technical route were raised according to their characteristics.Mechnical homogenate,ammonium carbonate extract,heat denaturation（60℃,10min）,pH 3.8 acetone fractional precipitation（or pH 4.6 acetone one time precipitation）,acetone washing for three times methods were used to extract the slow electrophoresis mobility DNases.Mechnical homogenate,ammonium carbonate extract, ammonium sulfate fractional precipitation,acetone washing methods were used to extract the fast electrophoresis mobility DNase.1.3 The Study on purification of a kind of low molecular weight Lumbricus Bimastus DNase（ELMWD） through chromatography and electrophoresis methodsDEAE anion-exchange chromatography,hydrophobic chromatography were used to purify ELMWD from Lumbricus Bimastus.Function gel and agar gel electrophoresis were used to monitor the active peak and collect them.Based on the chromatography purifiction,2D-electrophoresis and 2D- electrophoresis function gel were combined used to purify ELMWD.2.The study on the biochemistry characteristics and enzyme kinetics of ELMWDIn this section,SDS-PAGE was used to determine the MWs of ELMWD as 23KDa. Two-dimensional electrophoresis and capillary electrophoresis were used to measure the isoelectric point as pH 4.5.Ultraviolet spectrophotometry,agar gel electrophoresis were used to study the temperature optimization and stabilities,pH optimization and stabilities of the ELMWD.MALDI-TOF was used to study the purity,MWs,PMF.The result was that the optimum pH was 5.2,the optimum temperature was 40℃.The ELMWD was sensitive to the temperature.Different metal ions and substrates were studied on the effect of the ELMWD reaction velocity.Enzymological studies showed Kms and Vmax of ELMWD was 0.040μg/μl, 0.059μg/μl·min-1;0.090μg/μl,0.059μg/μl·min-1 when taking calf thymus DNA and plasmid DNA as substrates respectively.Mg²⁺,Ca²⁺,Mn²⁺,EDTA can inhibit the activity of ELMWD, however,5mM K⁺ can stimulate the activity.The MWs according to MALDI-TOF was 20KDa. The result of PMF and the parameters of ELMWD suggested that the ELMWD was a novel DNase.3.The study on the relationship between the DNase from Lumbricus Bimastus and the innate immunity of Lumbricus BimastusAccording to the innate immunity angle of degrading inside or outside abandoned cells and microbes,we carried out the study of earthworm coelomic fluid activity extraction action on virus,bacterium,tumor cells in vitro;At the same time,The study of tissue distribution of DNase in Lumbricus Bimastus has made us better understanding the function of the DNase.3.1 The tissue distribution of DNase and ELMWD in Lumbricus BimastusIimmunoblot,immunohistochemistry,SREB,function gel were used to detect the tissue distribution of ELMWD and DNase.Immunohistochemistry results showed that the DNaseE principally exists in the circular muscle of its body wall and the mucous membrane of intestinal wall outside.Immunoblot and activity measurement results showed that DNase and ELMWD mainly located in the coelomic fluid,coelomic fluid cell,intestinal.The results suggested that DNases have participated in the innate immunity and digestion of microbies.3.2 The study on the anti-bacterium activity of coelomic fluid activity extraction（ECF） and ELMWDThe in vitro anti-bacterium experiment was conducted by the degradation of structure of bacterium layer by layer.The Fluospectrophotometry method was used to test the effect of the ECF on the integrity of the cytoplasmic and outer membrane of E.coli.Spectrophotometric was used to test the effect on peptidoglycan from Gram-positive bacteria.Agarose gel electrophoresis was utilized to study the nucleases activities on the genome and plasmid of bacteria.The result showed that the the ECF have the ability of non-specific degradation of the bacteria structure from outside to inside.Agar gel electrophoresis and biofilm formation method were used to test the bacteriostasis mechanism of DNase from earthworm.The result showed that the DNase can kill the bacterium through degrading the genetic materials combined with other immunological moleculars and can inhibit the forming of biofilm.3.3 The study on the anti-tumor activity ECF and ELMWDThe MTT and SRB method were used to test the the ECF from Lumbricus Bimastus on the inhibition activity of four kinds of tumor cells.Hochest fluorescein stain and single cell gel electrophoresis（SCGE） were used to test the genomic condition of tumor cells.The result showed that the ECF possess the notable activity of inhibition the tumor growth;the cytogene were degraded to fragments.Apotosis was tested to exist through Flow cytometry.MTT method was further used to test the antineoplasmic activity of ELMWD.The result showed that the ELMWD had the inhibition effect of invasion and metastasis but not the inhibition of growth of tumor cells.3.4 The study on the anti-virus activity of ECF and ELMWDWe have assayed directly the virucidal effect on three kinds of virus（including DNA and RNA virus） by cell culture,Inhibition of virus-induced cytopathogenic effect.The conclusion was that the ECF possessed non-specific ability of degrading virus,while ELMWD has not significantly anti-viral activity but can Collaborative the ECFChromatography analysis indicated that the anti-viral components are mainly located in three active peaks that possess proteases,nucleases and lysozymes,respectively.In addition,three active fractions mixture had stronger antiviral activity than three fractions alone but lower than the ECF.Gel electrophoresis showed that nucleases from Lumbricus Bimastus can degrade the genome of virus.The ECF displayed non-specific antiviral properties and maybe mediated by the cooperation of proteases,nucleases and lysozymes.Conclusions:1.A new activity measurement was introduced which has high resolution and can monitor the change of DNase numbers during the purification.This has set a foundation for later studies.2.Two sets of DNases crude extract preparation technical routes for two groups of different electrophoresis mobility DNases were established.3.A mature ELMWD purification technical route was set up4.A kind of novel high activity DNase was purified.It’s molecular weight is 20759.417Da. It’s affinity with liner calf thymus DNA is higher than with the plasmid DNA.The optimum pH was 5.2;The optimum temperature was 37℃.It’s some enzymological kinetics characters and PMF result hint that this DNase is a kind of novel DNase different from others.5.Nuclease is one of the important elements of the innate immunity system.Nucleases play an important role in the degradation and inhibition microbes and abandoned cells in Lumbricus Bimastus independently or combined with other immune moleculars by degrading the genetic materials.It is significant to study and exploit the DNase from Lumbricus Bimastus.更多还原