【作者】 张建林；

【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】 目的1、分离纯化双胸蚓组织中的一种热稳定性核酸酶并对其进行特性研究2、克隆该热稳定性核酸酶的cDNA,推测其氨基酸序列并进行序列分析方法1、双胸蚓组织热稳定性核酸酶的分离和纯化以SDS-PAGE功能电泳法和紫外分光光度法作为双胸蚓组织热稳定性核酸酶提取过程中的活性监测方法。双胸蚓组织匀浆后通过热变性获得粗酶液;利用硫酸铵分级沉淀和80%丙酮沉淀获得核酸酶的粗品;粗品依次通过离子交换层析、疏水交换层析和凝胶过滤层析进行精细纯化。2、双胸蚓组织热稳定性核酸酶的表观特征和酶学性质研究以及杀菌杀病毒活性初探运用等电聚焦电泳、基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）和N-末端Edman技术等多种技术手段,确定双胸蚓组织热稳定性核酸酶的表观特征,如:分子量、等电点、肽质量指纹图谱（PMF）和N-末端氨基酸序列。通过不同条件下的活性对比实验,研究酶学特性,如:反应最适温度、最适pH、热酸碱稳定性等。以常见的几种病原微生物（包括细菌和病毒）为实验对象,采用琼脂铺板计数法和蚀斑减数法对其杀菌杀病毒活性进行初探。3、双胸蚓组织热稳定性核酸酶cDNA的克隆利用cDNA末端快速扩增（rapid-amplification of cDNA ends,RACE）技术进行cDNA克隆:提取双胸蚓组织总RNA,反转录得到cDNA,根据测定的N-末端氨基酸序列设计简并引物,作为上游扩增引物,ploy（A）通用引物作为下游扩增引物,扩增出双胸蚓组织热稳定性核酸酶的cDNA,与测序载体连接转化感受态细胞,筛选阳性克隆进行测序得到cDNA序列（含3’端非翻译区）,以此序列推导出氨基酸序列。4、双胸蚓组织热稳定性核酸酶cDNA序列和氨基酸序列的生物信息学分析根据获得的双胸蚓组织热稳定性核酸酶结构和功能信息,应用生物软件对其cDNA序列及氨基酸序列进行系统的分析和预测。内容包括核苷酸序列的碱基组成、密码子使用频率和酶切位点分析;氨基酸序列的氨基酸组成、疏水图谱和同源性比对;对蛋白质的分子量和等电点进行估算以及对二级结构和化学修饰位点进行分析预测。结果1、分离纯化出双胸蚓组织热稳定性核酸酶,纯度95%以上,浓度为0.28 mg/ml。2、测得双胸蚓组织热稳定性核酸酶精确分子量29897.322Da,等电点6.11。N-末端13个氨基酸为PLGPYKPKCDEWV。根据检测的肽质量指纹图谱（PMF）在蛋白质数据库中比对,没有检索到与之相匹配的任何蛋白,可能为一种未报道的新蛋白。3、通过对比实验确定了酶活性最适温度为25℃、最适pH值pH5.0-5.5之间;对热稳定,70℃保温60min仍具85%以上的残余活性; pH4.0-9.0之间核酸酶稳定性比较好;Mg²⁺、Mn²⁺、Ca²⁺对酶有激活作用,Zn²⁺、Fe²⁺、Cu²⁺对酶有抑制作用;对常用有机溶剂甲醇、乙醇和丙酮不敏感;单链线状DNA为双胸蚓组织热稳定性核酸酶最佳底物;以单链线状DNA为底物测得Km值为0.048mg/ml。4、双胸蚓组织热稳定性核酸酶对几种常见病原微生物:大肠埃希菌、金黄色葡萄球菌、白色念珠菌以及乙肝病毒和流感病毒都有不同程度的杀灭作用,其中对白色念珠菌和流感病毒杀灭作用相对较弱。5、克隆了双胸蚓组织热稳定性核酸酶的cDNA序列（含3’端非翻译区）。序列为864bp,包含一个729bp的开放阅读框ORF（1-729bp）,编码243个氨基酸。6、利用生物软件进行分析,获得cDNA序列碱基组成、密码子使用频率和酶切位点等详细数据以及蛋白质理论分子量、等电点和氨基酸组成等情况。蛋白稳定性分析结论为性质稳定。利用BLAST软件,将双胸蚓组织热稳定性核酸酶的蛋白序列与NCBI蛋白序列数据库比对,没有检索到与之相匹配的任何蛋白。二级结构分析获知蛋白主要由无规卷曲组成（约占50%）,5个α-螺旋中心（约占20%）,其余为20%左右为片层结构。结论1、成功纯化了一种双胸蚓组织热稳定性核酸酶。2、该核酸酶表观特征和酶学性质与已知的核酸酶有较大差异,对多种病原微生物具有不同程度的杀灭作用。3、成功克隆出该核酸酶cDNA序列。更多还原

【Abstract】 Object1. Separate and purify a thermostable nuclease from Lumbricus Bimastus and study on its properties.2. Clone the cDNA of the thermostable nuclease, deduce its amino acid sequence and analyze cDNA and amino acid sequence.Menthod1. Separation and purification of a thermostable nuclease from Lumbricus BimastusTake SDS-PAGE functional electrophoresis and UV spectrophotometry as activity monitoring methods. Obtain the crude enzyme solution by thermally denature earthworm tissue homogenate. Acquire the crude enzyme by ammonium sulfate fractional precipitation and 80% acetone precipitation. Purify the crude enzyme by ion exchange, hydrophobic exchange and gel filtration chromatography.2. Characterization of the nature and enzymatic properties of thermostable nuclease from Lumbricus Bimastus and determination of the killing activity against Bacteria and virusesUse isoelectric focusing electrophoresis（IEF）、matrix-assisted laser desorption time of flight mass spectrometry （MALDI-TOF-MS）、N-terminal Edman technique and other technology means to research the apparent characteristics, such as: Molecular weight, isoelectric point, peptide mass fingerprinting （PMF）, N-terminal amino acid sequence. Through Comparative experiment under different conditions study on enzyme characteristics, such as: reaction optimum temperature, optimum pH, hot and acid-base stability. Take several common pathogenic microorganisms （including bacteria and viruses） as the experimental object to investigate its antifungal and antiviral activity using agar plate colony counting and plaque counting assay.3. cDNA cloning of the thermostable nuclease from Lumbricus BimastusEmploy rapid amplification of cDNA ends （RACE） technology for cDNA cloning, Process is as follows: extracted total RNA, reverse transcription obtained cDNA and designed mergers primers, as the upstream primer, according to the determination of the N-terminal amino acid sequence, taked ploy （A） universal primer as the downstream primer.Amplified the cDNA of the thermostable nuclease, Recovered PCR product by Cutting Gel. Connected sequencing vector, transformed into competent cells, followed by selected positive clones have been sequenced cDNA sequences （including 3 ’UTR） and deduced the amino acid sequence.4. Analysis cDNA sequence and amino acid sequence of the thermostable nuclease from Lumbricus Bimastus with bioinformatics methodsAccording to the information of structure and function, analysis cDNA sequence and amino acid sequence of the thermostable nuclease from Lumbricus Bimastus with bioinformatics methods. Include: the base composition of nucleotide sequence, codon usage and restriction site analysis; amino acid sequence composition, the hydrophobic pattern and homology comparison; estimate protein molecular weight and isoelectric point as well as secondary structure prediction and chemical modification sitesResult1. Separated and purified a thermostable nuclease from Lumbricus Bimastus, more than 95% purity, concentration is 0.28 mg/ml.2. Measured enzyme precise molecular weight is 29897.322Da and isoelectric point is 6.11. N-terminal 13 amino acids are PLGPYKPKCDEWV. According to comparison peptide mass fingerprinting （PMF） in the protein database, there is no search to match any protein, may be a new unreported protein.3. Through comparative experiment under different conditions, identified the optimal activity temperature is 25℃, the optimum pH value between pH5.0-5.5; of heat-stable, 70℃insulation 60min still have more than 85% residual activity; nuclease is stable between pH5.0-5.5 ,Mg²⁺, Mn²⁺, Ca²⁺ has activation effect on the enzyme, Zn²⁺, Fe²⁺, Cu²⁺ has inhibitory effect on the enzyme; not sensitive to acetone and ethanol of common organic solvents methanol. Linear DNA is best substrate to the thermostable nuclease from Lumbricus Bimastus; Km value is 0.048mg/ml with single-stranded linear DNA as substrate.4. The thermostable nuclease from Lumbricus Bimastus has fungicidal effect against Escherichia, Staphylococcus, Candida albicans,hepatitis b virus and influenza virus in different extent. The fungicidal effects against Candida albicans and influenza virus are relatively weak.5. Cloned the cDNA of thermostable nuclease from Lumbricus Bimastus, which containing 3- ’UTR of the 864bp cDNA sequence, contains a 729bp of the open reading frame ORF （1-729bp）, encoding 243 amino acids6. With biological software analysis, obtained cDNA sequence base composition, codon usage and restriction site data, as well as detailed theoretical protein molecular weight, isoelectric point and amino acid composition. Protein stability analysis concluded that the nature of stability.Using BLAST software, compared amino acid sequences of thermostable nuclease from Lumbricus Bimastus with the sequences of protein sequence database, homology search to the very poor did not match any protein. Protein secondary structure was informed mainly by the random coil component （about 50%）, 5α-helix centers （about 20%）, and the remaining 20% of the lamellar structure by biological software analysis Conclusion1. Successfully purifid a nuclease from Lumbricus Bimastus, characteristics study has confirmed its high thermal stability.2. The nature and enzymatic properties of thermostable nuclease from Lumbricus Bimastus is quite different form known nuclease. It is able to kill a variety of pathogenic microorganisms with different levels.3. Successfully cloned cDNA fragment of the thermostable nuclease from Lumbricus Bimastus更多还原