导　　师： 庞辉

学科专业： 071003

授予学位： 硕士

作　　者： ;

机构地区： 广西医科大学

摘　　要： 目的从螺旋藻发酵物中分离纯化出螺旋藻激酶,通过质谱分析获得螺旋藻激酶的蛋白相关信息,为日后基因工程生产螺旋藻激酶和进一步研究其结构和功能提供理论依据。 方法/(1/)制备螺旋藻激酶粗提液：螺旋藻发酵物通过水提法,再通过切向超滤系统超滤后获得螺旋藻激酶粗提液,选用考马斯亮蓝G-250染色法测定其蛋白含量；/(2/)螺旋藻激酶粗提液经SephadexG-75凝胶过滤层析进一步纯化,将获得的洗脱峰用纤维蛋白平板实验筛选出有溶栓活性的洗脱峰；/(3/)将有溶栓活性洗脱峰浓缩,进行SDS-PAGE电泳；/(4/)选用MADLI-TOF-TOF-MS质谱分析纯化的螺旋藻激酶的蛋白相关信息。 结果1、螺旋藻激酶的分离纯化结果/(1/)螺旋藻发酵物通过水提法,再通过切向超滤系统超滤后获得螺旋藻激酶粗提液,考马斯亮蓝G-250染色法测定螺旋藻激酶粗提液的蛋白含量是1.735ug//ml。/(2/) Sephadex G-75凝胶过滤层析螺旋藻激酶粗提液经SephadexG-75凝胶过滤法分离,得到5个洗脱峰,以尿激酶为阳性对照组,纤维蛋白平板法检测各洗脱峰溶栓活性,其中只有第5个洗脱峰有明显的裂解圈,显示出溶栓活性,收集此峰洗脱液。/(3/) SDS-PAGE电泳结果Sephadex G-75凝胶过滤法分离得到5个洗脱峰,选用唯一有溶栓活性的第5个洗脱峰,SDS-PAGE电泳得到单一螺旋藻激酶蛋白条带,参照蛋白标准物,可知螺旋藻激酶的分子量约为45KD。2、螺旋藻激酶的蛋白相关信息：/(1/)编号为gi｜493675806的一级质谱报告,显示犬尿氨酸甲酰胺酶分子量为43705道尔顿,等电点为4.91,提示螺旋藻激酶的分子量接近43705道尔顿。质谱鉴定结果表明螺旋藻激酶固定的氨基酸修饰是C-端乙酰胺化,可变的氨基酸修饰是蛋白N端乙酰化、去酰胺化、二氧化物氧化。/(2/)编号为gi｜493675806的二级质谱报告,显示将待鉴定的纯化蛋白条带凝胶用胰蛋白酶酶切后,肽段混合物上MALDI-TOF-TOF,螺旋藻激酶纯化蛋白IYFPVYVEGAK肽段相对分子量是1284.6754,其观测值与理论值的偏差是百万分之121,该肽段相对于整个蛋白质的得分是76,氨基酸修饰位点是脲甲基化。/(3/)生物信息学分析结果表明螺旋藻激酶的氨基酸序列与蓝藻目极大节螺旋藻犬尿氨酸甲酰胺酶有较高相似度,蛋白覆盖率为35/%,提示两者有同源性。 结论从螺旋藻发酵物中成功纯化出电泳纯的单一条带的螺旋藻激酶,MADLI-TOF-TOF-MS质谱分析获得其蛋白相关信息,说明了螺旋藻激酶和犬尿氨酸甲酰胺酶有同源性,分子量接近43705道尔顿,分别约是纳豆激酶和豆豉纤溶酶分子量的1.5倍,螺旋藻激酶是一种有别于纳豆激酶和豆豉纤溶酶的新型纤溶酶。 目的：通过体外培养野生型MEF细胞和突变型Zmpste24-//-MEF细胞,探讨螺旋藻激酶粗提液对野生型MEF细胞和突变型Zmpste24-//-MEF细胞的细胞形态、细胞活力及衰老细胞阳性率的影响,从抗衰老药物功效方面探讨螺旋藻激酶粗提液对野生型MEF细胞和突变型Zmpste24-//-MEF细胞的延缓细胞衰老作用,为开发新型抗衰老药物提供理论依据。方法：1、细胞株制备：从正常孕鼠怀孕13.5天后取小鼠胚胎成纤维细胞,传代培养,建立野生型MEF细胞模型；从Zmpste24+//-基因敲除杂合子孕鼠怀孕13.5天后取Zmpste24-//-基因敲除小鼠胚胎成纤维细胞,传代培养,建立突变型Zmpste24-//-MEF细胞模型。2、实验分组：实验分组为空白对照组和给药组,空白对照组培养过程中用10/%PBS+细胞生长液；给药组培养过程中用10/%PBS+细胞生长液+10/%螺旋藻激酶粗提液/(0.036mg//ml/),研究螺旋藻激酶粗提液对野生型MEF细胞和突变型Zmpste24-//-MEF细胞的抗衰老作用。3、检测项目：/(1/)将螺旋藻激酶粗提液作用于突变型Zmpste24-//-MEF细胞,显微镜下观察其细胞形态学的变化；/(2/)CCK-8法检测螺旋藻激酶粗提液影响野生型MEF细胞和突变型Zmpste24-//-MEF细胞的增殖情况并绘制细胞活力曲线；/(3/)p-半乳糖苷酶染色法检测螺旋藻激酶粗提液影响野生型MEF细胞复制性衰老细胞阳性率。 结果：1、螺旋藻激酶粗提液影响突变型Zmpste24-//-MEF细胞的细胞形态学变化：空白对照组细胞排列不规则,体积较大形态扁平,胞内分泌型颗粒明显增多,胞质透明度低,部分核仁胀大破裂,核内颗粒释放。给药组生长良好,形态正常,呈长梭形或不规则形,细胞内分泌型颗粒极少,胞质透明度较大,核仁完整饱满、轮廓清晰,细胞呈河流状或放射状生长。2、螺旋藻激酶粗提液影响野生型MEF细胞和突变型Zmpste24-//-MEF细胞的增殖情况：从细胞活力曲线可见,正常孕鼠怀孕13.5天后取到的小鼠胚胎中培养的野生型MEF细胞,螺旋藻激酶粗提液作用后,CCK-8法测得其细胞活力在0-144h期间的96h和120h处理时间点分别为106.46/%和129.03/%/(P/(?/)0.05/),而120h处理时间点细胞活力最高；表明了在120h处理时间点的活细胞数目最多,细胞生长状态最好,对野生型MEF细胞影响最大。说明螺旋藻激酶粗提液对野生型MEF细胞起到延缓细胞衰老的作用。螺旋藻激酶粗提液对突变型Zmpets24-//-MEF细胞的细胞活力数据经过医学统计学方法分析,P/(?/)0.05,无统计学意义,说明螺旋藻激酶粗提液对突变型Zmpets24-//-MEF细胞无延缓野生型MEF细胞衰老作用。3、p-半乳糖苷酶染色法检测野生型MEF细胞衰老细胞阳性率：野生型MEF细胞空白对照组细胞染色阳性率为20.85/%,给药组细胞染色阳性率为8.74/%/(P<0.05/)。结论：1、螺旋藻粗提液可以维持突变型Zmpste24-//-MEF细胞的年轻态。2、螺旋藻激酶粗提液对野生型MEF细胞起到延缓细胞衰老的作用。3、螺旋藻激酶粗提液下调了p-半乳糖苷酶的表达活性,抑制细胞衰老,延长细胞寿命,促进了细胞的更新与增值。 Abstract Objective:To purify Spirulinakinase from crude fermentative Spirulina and analyze the information on protein of Spirulinakinase using mass spectrometry analysis, providing experimental basis for producing genetic engineering spirulinakinase and studying its structure and function further. METHODS:/(1/) Spirulinakinase crude extract was prepared by water extraction and was ultrafiltrated by the tangential ultrafiltration system, and its protein content was determined by CBB G-250/(Coomassie brilliant blue G-250/) staining./(2/) Spirulinakinase crude extract were got by Sephadex G-75gel filtration chromatography futher purification. The filbrinolytic activity was screened by fibrin plate experiment./(3/) The strip of spirulinakinase was got by SDS-PAGE electrophoresis after the concentration of the elution peak which had the fibrinolytic activity./(4/) The information on protein of spirulinakinase was analyzed by MADLI-TOF-TOF-MS spectrometry. RESULT:The separation and purification of Spirulinakinase:/(1/) Spirulinakinase crude extract was prepared by water extraction and ultrafiltrated by the tangential ultrafiltration system, and its protein content was determine by CBB G-250/(Coomassie brilliant blue G-250/) staining, and the concent of spirulinakinase protein was1.735ug//ml./(2/) Sephadex G-75gel filtration chromatography:Five elution peaks were got by separating spirulinakinase crude fermentation ultrafiltrate through Sephadex G-75gel filtration chromatography. The fibrinolytic activity of the fifth elution peak was determined by fibrin plate method with urokinase as positive group, and the obvious cracking ring was shown to improve thrombilysis activity, and the fifth elution peak was collected./(3/) SDS-PAGE electrophoresis result:Five elution peaks were got by Sephadex G-75gel filtration separation,and the fifth peak which had thrombolysis activity was determined by SDS-PAGE electrophoresis and the single protein strip was obtained to show the molecular weight as45KD consulting standard protein content.2.The analysis on the information of spirulinakinase protein:/(1/) The result that molecular weight43705Dalton of urine glycine formamide and isoelectric point4.91were shown on the Mass report No.gi493675806. And the molecular weight of spirulinakinase was prompted to be close to43705Dalton. And the result of Mass spectrum identification that the fixed modification amino acids of spirulinakinase C-end acetamide, variable amino acid modified protein N acetyl,amide and dioxide oxidation were shown on the Mass report./(2/) The result that the neutral peptides single isotope quality theoretical value1284.6754of spirulinakinase protein purification IYFPVYVEGAK peptides was shown on Mass report No.gi1493675806through protein which would had been purified and idetificated by trypsin banding gel with enzyme digestion and peptides mixture was determined by MALDI-TOF-TOF-MS. The observation value with the theoretical value of deviation was on hundred and twenty-one over one million,the peptide score was76relative to the entire protein amino acid and the modified site was urea methylation./(3/) The result of bioinformatics analysis that the high similarity of protein coverage rate of35/%between the amino acid sequence of Arthrospira maxima and urine glycine fomamide enzymes. And that was suggested they were homologous. Conclusion:The strip of spirulinakinase was purified from crude fermentative spirulina successfully, and its information that the homology between spirulinakinase and urine glycine formamide enzymes was confirmed by MADLI-TOF-TOF-MS which was used to get information about Spirulinakinase protein. And the difference between molecular weight43705Dalton of spirulinakinase and that of natto kinase or douche fibrinolytic enzyme was suggested that spirulinakinase was a new type fibrinolytic enzyme which was different from natto kinase and douche fibrinolytic enzyme. OBJECTIVE:To study on the effect of spirulinakinase crude extracting solution about cell morphology, cell activity and senescent cells positive influence by culturing wild type MEF cells in vitro, in order to discusse the effect of spirulinakinase on cells senescence about two types that wild type MEF cells and mutant Zmpste24-//-MEF cells from the aspects of anti-aging efficacy of spirulinakinase extracting solution,providing theoretical basis for developing new anti-aging drugs. METHODS:1、 Cell model building:Wild type MEF cells model was subcultured and set up from the normal pregnant rats after13.5days pregnancy. And mutant Zmpets24-//-MEF cells model was subcultured and set up from the Zmpets24+//-knockout heterozygous pregnant rats after13.5days pregnancy.2、Groups of experiment:Groups of experiment were divided into two parts,one was the blank control group with the solution of10/%PBS and cells growth liquid, the other group was drugs control group withthe solution of10/%PBS,cells growth liquid and10/%Spirulinakinase crude extracting solution /(0.036mg//ml/), in order to research anti-aging effect of Spirulinakinase crude etracting solution on two cells types that wild type MEF cells and mutant Zmpets24-//-MEF cells.3、Testing items:/(1/) The change of the mutant24-//-MEF cells morphology was observed under a microscope./(2/) The proliferation of wild type MEF cells and mutant24-//-MEF cells were tested by CCK-8method./(3/) The cell positive rate of replication senescence of wild type MEF cells was detected by β-galactose glucoside enzyme staining method. RESULTS:1、Cells morphological changes of mutant Zmpets24-//-MEF cells:Cells blank control was observed with situation that cells arrangement was irregular,cells form was large and flat, cells endocrine particles increased obviously, cytoplasmic transparency was low,part of the nucleolus swell was broken and particles in nuclear released.On the contrary, drugs control was observed with situation that cells grew well,configuration was normal,cells form was a long fusiform or irregular shape,endocrine cells had few particles,cytoplasmic had a great transparency,nucleous was full, outline was clear, cells grew as a riverlike shape or radial.2、The proliferation of wild type MEF cells and mutant Zmpets24-//-MEF cells:From the cells vitality curve, wild type MEF cells from the mouse of the normal pregnant rats after13.5days pregnancy were treated by spirulinakinase crude extracting solution, and then were measured by CCK-8staining method,and the cells vitality106.46/%/(P/(?/)0.05/) at96h processing time point and129.03/%/(P/(?/)0.05/) at120h processing time point were determined during0-144h. And the maximum was the value of120h processing time point. The result was shown that the cells anti-aging effect of spirulinakinase crude extracting solution was obvious on wild type MEF cells. With comparation, the effect of anti-aging on mutant Zmpets24-//-MEF cells was negative because of the P value higher than0.05./(3/) Cells senescence was detected by β-gal staning method:The positive rate of blank control was20.85/%, and positive rate of drug group was8.74/%. CONCLUSION:1、The growth state of wild type was better due to the reason that the young state of the mutant Zmpets24-//-MEF cells was maintained with spirulinakinase effect.2、The result of experiment CCK-8stainning that the biggest number and the best statue of cells growth of wild type MEF cells which was treated by drug spirulinakinase was at the processing time120h compared to that of the processing time0h because of anti-aging effect of spirulinakinase.3、The result of experiment β-gal stainning that the positive rate of drug control was lower than that of blank control was due to the reason that the activity of β-gal enzyme was downgraded, the cells aging was inhibited, the life of cells was prolonged, and the renewal of cells and appreciation were promoted.

关 键 词： 螺旋藻激酶 分离纯化 质谱分析 纤溶活性 细胞衰老 半乳糖苷酶染色 细胞生长曲线 细胞活力 细胞形态学 小鼠胚胎成纤维细胞

分 类 号： [Q946.5]

领　　域： [生物学]