【作者】 魏文志；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学与工程， 2008， 博士

【摘要】 小球藻是一种人工培养的营养保健品,含有多种营养成分。近年来的研究表明,小球藻热水抽提物和干粉具有抗肿瘤及其它多种生物功能,其中起主要作用的是糖蛋白提取物。但目前的研究主要集中在糖蛋白粗提物及其在动物的体内试验,对小球藻糖蛋白的分离纯化和预防肿瘤作用报道甚少。同时传统的肿瘤药物筛选方法是针对疾病或者针对化合物,只适合于随机筛选。本论文对小球藻糖蛋白进行了分离纯化,并根据肿瘤预防的机理建立一个快速、灵敏、高通量而又经济的体外细胞筛选模型,对小球藻糖蛋白预防肿瘤的作用进行了筛选,不仅对小球藻预防肿瘤药物的开发利用具有一定的理论意义和应用价值,同时也为筛选利用其它天然药物提供了借鉴作用。首先对小球藻糖蛋白进行了分离纯化。运用响应面分析确定了小球藻糖蛋白水提法的最佳提取条什:料水比1:21.9,在温度86.0℃下提取5.0 h;运用正交试验确定了Sevag法的最佳脱蛋白条件:样液与试剂比2:1,氯仿与正丁醇比4:1,脱蛋白次数3次,脱蛋白时间15 min;经Sephadex-75层析进一步纯化、透析、浓缩、冷冻干燥后,得到两种白色棉花状糖蛋白物质（CGPⅠ和CGPⅡ）。SephadexG-200凝胶柱层析证明CGPⅠ为均一组分,CGPⅡ组分不均一,高压凝胶过滤色谱证实CGPⅠ纯度为98.35%,CGPⅡ的纯度为78.84%。对小球藻糖蛋白CGPⅠ的化学组成进行了研究。通过SDS-PAGE,显示CGPⅠ呈现一条带,相对分子质量为63,700:氨基酸自动分析仪测定CGPⅠ含有全部17种氨基酸,并富含酸性氨基酸。气相色谱显示CGPⅠ主要单糖组成为葡萄糖、半乳糖、鼠李糖。紫外可见光谱分析显示CGPⅠ有蛋白质和糖吸收峰,没有核酸吸收峰;红外光谱分析表明CGPⅠ的糖苷类型为吡喃型。β-消去反应说明CGPⅠ糖组分和蛋白质组分是通过0-糖肽键连接的。测定了小球藻糖蛋白CGP I的体外抗氧化活性。通过清除羟自由基、超氧阴离子自由基、DPPH自由基、烷基自由基、还原力等试验,测定小球藻糖蛋白的抗氧化活性。结果显示,不同浓度CGPⅠ有一定的还原能力,且具有清除羟自由基、超氧阴离子自由基、DPPH自由基、烷基自由基的能力,其还原能力和清除自由基的能力与剂量有一定的效应关系。CGPⅠ消除羟自由基的能力高于Vc,其还原能力、清除超氧阴离子自由基、DPPH自由基、烷基自由基的能力均低于Vc。建立了体外筛选化学预防剂的转基因细胞模型筛选小球藻糖蛋白的肿瘤预防作用。应用重组PCR技术,从pRL-TK上扩增重组胸腺嘧啶核苷激酶（TK）启动子,并在其基本启动子上游创建特异性酶切位点SacⅠ。第一轮PCR获得500 bp和150 bp两个片段,第二轮PCR获得635bp的目的片段,克隆入pMD18-T载体中。而后扩增TK基本启动子克隆入pEGFP中构建载体pTK-GFP,并在其上游插入抗氧化反应元件（ARE）片段,构建pARE-TK-GFP。最后从以上两载体中扩增TK和ARE-TK目的片段克隆入载体pEGFP-N₁中,构建最终表达载体pTK-GFP/neo和pARE-TK-GFF/neo。两表达载体分别用脂质体法转染HepG2细胞后,用800μg/ml G418筛选出细胞克隆HepG2-TK-GFP和HepG2-ARE-TK-GFP,在荧光显微镜下可见明显的绿色荧光。将阳性克隆细胞扩大培养,加入糖蛋白溶液,使其终浓度分别为0.32、1.6、8、40、200（μg/ml）,同时用PDTC和香菇多糖作阳性对照。结果显示,糖蛋白在200μg/ml时诱导效果最好,且糖蛋白浓度与GFP相对荧光强度存在剂量效应关系,而在对照克隆细胞中未发现与受试物有剂量效应关系。本研究从小球藻中分离得到一种糖蛋白,该糖蛋白具有抗氧化活性;根据肿瘤预防的抗氧化机理构建了一个带有抗氧化反应元件（ARE）和绿色荧光蛋白基因（GFP）载体的转基因细胞模型;小球藻糖蛋白能诱导ARE的顺式表达作用,促进GFP的荧光强度,并有剂量的依赖关系,说明小球藻糖蛋白具有预防肿瘤的作用。更多还原

【Abstract】 Cklorella pyrenoidosa is man-cultured health supplement contained kinds of compounds.The hot water extracts(CVE) and power of Chlorella pyrenoidosa have been reported to possess various pharmacological effects including antitumor activities and potent biological response modifiers that modulate immune responses against bacteria and viruses,and it mainly is the glycoprotein extracts that play an important role.The study on Chlorella pyrenoidosa focused on the extracts and its functions in vivo,but the report on the and tumor-prevention is rare.Traditional methods of screening the drug for antienoplastic in the light of dieases and compounds suited only to random screening.In order to use this resource,the isolation,purifition of glycoprotein from Chlorella pyrenoidosa were carried and a rapid,sensitive,high throughout and cheap screening method in vitro was constructed to study the tumor-prevention of glycoprotein from Chlorella pyrenoidosa.It will be of scientific significance in researching and developing the new synthesized and natural tumor-prevention.The glycoprotein from Chlorella pyrenoidosa were isolated and purifed firstly. The technology and condition of glycoprotein extraction from Chlorella pyrenoidosa were studied.Water extraction was used to extract glycoprotein,the optimum extraction conditions of glycoprotein were chosen by considering the effect of each factor and subsequently doing response surface experiment.The better condition of glycoprotein extraction is:the ratio of raw material to water is 1:21.9,extracting temperature is 86.0℃and extracting time is 5.0 h;Sevag method was used to remove protein.The optimum conditions was chosen by considering the effect of each factor and subsequently doing orthogonal experiment,and the better condition of removing protein is:the ratio of sample liquid to Sevag liquid is 2,the ratio of chloroform to 1-butanol is 4,removing protein times is 3,the time of removing protein is 15 min.The crude glycoprotein was further purified by Sephadex G75 gel,dialysis, concentration.After vacuum freeze-drying,two white flocculent purified glycolproteins were obtained.Elution patterns on sephadexG-200 column chromatography proven CGP I was homogeneous and CGPⅡwas not homogeneous.The results of HPLC indicated the purity of CGPⅠand CGPⅡwere 98.35%and 78.84% respectively.Chemical composition of CGPⅠwas studied.The results of SDS-PAGE of CGPⅠshowed a single spot and the molelular weight is 63,700;CGPⅠcontains 17 amino acids and two acidic amino acids(Asp and Glu) is higher using amino acid analyzer;Lack of absorption at 260.00nm by UV scaning indicated that CGPⅠcontained no nucleic acid.It was proved that the main monosacchrides were glucose, galactose and xylose by gas chromatography.The type of the glucosidic bonds was pyranose by IR and O-glycosidic linkage was found byβ-elimination reaction.The antioxidant activities in vitro of glycoprotein from Chlorella pyrenoidosa were researched through determinations of reducing power and scavenging hydroxyl free radical,superoxide free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical,the results showed that the different concentrations CGPⅠhad stronger reducing power and scavenging effect on superoxide free radical,hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical in a concentration of dose-dependant manner.The scavenging capacity of CGPⅠin the hydroxyl free radical was stronger than Vc,but the scavenging capacity to superoxide free radical, hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical was lower than Vc.A transgenic cell model was established to screen preliminarily in vitro the tumor prevention of glycoprotein from Chlorella pyrenoidosa.The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the SacⅠenzyme site was added.Then the 500 bp and 150 bp segments obtained from the first round of the recombinant PCR and the 635 bp segment from the second round PCR were both identified then cloned into pMD18-T vector.The TK promoter was amplified from it and cloned into pEGFP to construct pTK-GFP,the ARE enhancer was inserted into the upstream of TK to construct the vector pARE-TK-GFP.At last the TK and ARE-TK segments were amplified and cloned into pEGFP-N1 so the eukaryotic expression vectors pTK-GFP/Neo and pARE-TK-GFP/Neo were constructed.The two vectors were transfected into HepG2 cells and clones resistant to 800μg/ml G418 were isolated and named as HepG2-TK-GFP and HepG2-ARE-TK- GFP of which the green fluorescence was obvious under the fluorescent microscope.The different concentration(0.32,1.6,8,40,200μg/ml)of glycoprotein was added into the transgenic cells and the PDTC and lentinan were as controls.The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dosedependency with the different concentrations of glycoprotein in a certain range while the controls have not.In the study,a glycoprotein from Chlorella pyrenoidosa was obtained,and it anti-oxidation activities was proved;According to the the antioxidant mechanism of tumor-prevention,A green fluorescent protein(GFP) transgenic cell model under the transcriptional control of TK promoter and ARE was constructed to evaluate the tumor-preventive activity of glycoprotein from Chlorella pyrenoidosa.The glycoprotein could induce the cis-expression of ARE and promote the fluorescence intensity of GFP,and the induced level of GFP have dose-dependency with the concentrations of glycoprotein.So,the tumor-prevention of the glycoprotein from Chlorella pyrenoidosa was made sure preliminarily.更多还原