构建转基因细胞模型筛选小球藻糖蛋白预防肿瘤作用的研究
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摘要
本文在国内首次报道了从小球藻中提取的一种糖蛋白具有预防肿瘤作用。首先通过热水浸提、脱蛋白、醇析等步骤从小球藻中分离出一种糖蛋白,并对提取工艺中的料水比、温度、提取时间、提取次数等单因素进行优化以达到最佳提取效果。其次机体内的Ⅱ相酶在防治肿瘤发生过程中起着非常重要的作用,Ⅱ相酶基因启动区有一保守的DNA基序被称为抗氧化反应元件(ARE),可调控下游相关基因表达。据此原理,以TK为基本启动子,将人工合成的ARE与绿色荧光蛋白(GFP)基因相串联,构建ARE调控的报告载体pARE-TK- GFP/neo和对照载体pTK-GFP/neo。分别转染HepG2细胞用G418筛选出阳性克隆。然后在阳性细胞中加入小球藻糖蛋白溶液,作用48h后检测细胞的相对荧光强度,从而表明其对肿瘤的化学预防效果。具体研究内容及结果如下:
1.对小球藻糖蛋白提取工艺中的各单因素进行正交实验,确定了小球藻糖蛋白的最佳提取条件为:料水比1:20,在温度70℃下提取2次,每次6h。在此条件下糖组分得率最高。对所提取的糖蛋白进行凝胶柱层析鉴定和SDS-PAGE电泳鉴定,结果表明其为电泳纯单一糖蛋白,据蛋白质标准曲线可得出该糖蛋白分子量约为63.7KDa。
2.应用重组PCR技术,从pRL-TK上扩增重组TK启动子,并在其基本启动子上游创建特异性酶切位点Sac I。第一轮PCR获得500 bp和150 bp两个片段,第二轮PCR获得635 bp的重组TK启动子,经电泳初步鉴定正确后克隆入pMD18-T载体中又经酶切鉴定正确。扩增后的重组TK启动子克隆入pEGFP中构建载体pTK-GFP,并在其上游插入ARE片段构建pARE-TK-GFP,两者均经PCR鉴定正确。最后从以上两载体中分别扩增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-GFP/neo,酶切和测序鉴定结果表明目的片段插入位置正确且无突变。
3.两表达载体分别用脂质体法转染HepG2细胞后,用800μg/ml G418筛选出阳性细胞克隆HepG2-TK-GFP和HepG2-ARE-TK-GFP,在荧光显微镜下可见明显的绿色荧光。阳性克隆细胞经扩大培养后加入不同浓度梯度的糖蛋白溶液,同时用已知化学预防剂PDTC和香菇多糖作对照。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相对荧光度间存在剂量效应关系,而在对照细胞中未发现与受试物有剂量效应关系。
本实验构建了一个可用于体外快速筛选诱导Ⅱ相酶化合物的转基因细胞模型,为各种天然或人工合成具有预防肿瘤作用化合物的筛选奠定了基础,同时也可为小球藻糖蛋白作为肿瘤预防剂的开发提供了初步的实验依据。
It is the first time to report that the glycoprotein extract from Chlorella pyrenoidosa have chemopreventive activity in china. The crude glycoprotein was extracted with hot water and precipitated by ethanol after deproteinization, the best extraction process and conditions were chosen by optimize the effect of four factors as follows: the ratio of raw material to water, temperature, extracting time and extracting times. Because the phaseⅡenzyme play an important role in preventing cancer , the promoter of the enzyme gene has DNA motifs called anti-oxygen response elements (ARE) which are very conservative and can regulate the expression of the down stream genes. Based on this principle, we constructed the eukaryotic GFP reporter vector pARE-TK-GFP/neo under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted and the control vector pTK-GFP/neo. The two vectors were transfected into HepG2 cells and clones resistant G418 were isolated. After that the different concentrations of glycoprotein were added into the 96-well plate in which transgenic cells were seeded, 48h later the different induced levels of GFP were measured to indicate the effect of the glycoprotein. The results were as follows:
1. The optimum extraction process and conditions were chosen by considering the effect of each factor and subsequently doing orthogonal experiment: the ratio of raw material to water is 1:20, extract two times under the temperature of 70℃and each times with 6h. This is the best condition to extract glycoprotein because the obtained sugar components were the highest. The homogeneity was examined with Sephadex-200 column chromatography and SDS-PAGE, and the molecular weight was established to be about 63.7kDa on SDS-PAGE.
2. The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the Sac I enzyme site was added. The 500bp and 150bp segments obtained from the first round of the recombinant PCR, the 635bp segment from the second round PCR were 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, they were both identified by PCR. 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, which were identified by enzyme cutting and sequencing.
3. 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. The green fluorescence was obvious under the fluorescent microscope. The different concentration of glycoprotein was added into the transgenic cells and the known chemopreventive agents PDTC and lentinan were as controls. The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dose-dependency with the different concentrations of glycoprotein in a certain range while the controls have not.
This study primary constructed transgenic cell model which can induce the phaseⅡenzyme. It was not only provides a basis for the screening of natural or synthetic cancer chemopreventive agents, but also provides experimental evidence for the Chlorella pyrenoidosa developed as chemopreventive agents.
1.对小球藻糖蛋白提取工艺中的各单因素进行正交实验,确定了小球藻糖蛋白的最佳提取条件为:料水比1:20,在温度70℃下提取2次,每次6h。在此条件下糖组分得率最高。对所提取的糖蛋白进行凝胶柱层析鉴定和SDS-PAGE电泳鉴定,结果表明其为电泳纯单一糖蛋白,据蛋白质标准曲线可得出该糖蛋白分子量约为63.7KDa。
2.应用重组PCR技术,从pRL-TK上扩增重组TK启动子,并在其基本启动子上游创建特异性酶切位点Sac I。第一轮PCR获得500 bp和150 bp两个片段,第二轮PCR获得635 bp的重组TK启动子,经电泳初步鉴定正确后克隆入pMD18-T载体中又经酶切鉴定正确。扩增后的重组TK启动子克隆入pEGFP中构建载体pTK-GFP,并在其上游插入ARE片段构建pARE-TK-GFP,两者均经PCR鉴定正确。最后从以上两载体中分别扩增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-GFP/neo,酶切和测序鉴定结果表明目的片段插入位置正确且无突变。
3.两表达载体分别用脂质体法转染HepG2细胞后,用800μg/ml G418筛选出阳性细胞克隆HepG2-TK-GFP和HepG2-ARE-TK-GFP,在荧光显微镜下可见明显的绿色荧光。阳性克隆细胞经扩大培养后加入不同浓度梯度的糖蛋白溶液,同时用已知化学预防剂PDTC和香菇多糖作对照。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相对荧光度间存在剂量效应关系,而在对照细胞中未发现与受试物有剂量效应关系。
本实验构建了一个可用于体外快速筛选诱导Ⅱ相酶化合物的转基因细胞模型,为各种天然或人工合成具有预防肿瘤作用化合物的筛选奠定了基础,同时也可为小球藻糖蛋白作为肿瘤预防剂的开发提供了初步的实验依据。
It is the first time to report that the glycoprotein extract from Chlorella pyrenoidosa have chemopreventive activity in china. The crude glycoprotein was extracted with hot water and precipitated by ethanol after deproteinization, the best extraction process and conditions were chosen by optimize the effect of four factors as follows: the ratio of raw material to water, temperature, extracting time and extracting times. Because the phaseⅡenzyme play an important role in preventing cancer , the promoter of the enzyme gene has DNA motifs called anti-oxygen response elements (ARE) which are very conservative and can regulate the expression of the down stream genes. Based on this principle, we constructed the eukaryotic GFP reporter vector pARE-TK-GFP/neo under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted and the control vector pTK-GFP/neo. The two vectors were transfected into HepG2 cells and clones resistant G418 were isolated. After that the different concentrations of glycoprotein were added into the 96-well plate in which transgenic cells were seeded, 48h later the different induced levels of GFP were measured to indicate the effect of the glycoprotein. The results were as follows:
1. The optimum extraction process and conditions were chosen by considering the effect of each factor and subsequently doing orthogonal experiment: the ratio of raw material to water is 1:20, extract two times under the temperature of 70℃and each times with 6h. This is the best condition to extract glycoprotein because the obtained sugar components were the highest. The homogeneity was examined with Sephadex-200 column chromatography and SDS-PAGE, and the molecular weight was established to be about 63.7kDa on SDS-PAGE.
2. The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the Sac I enzyme site was added. The 500bp and 150bp segments obtained from the first round of the recombinant PCR, the 635bp segment from the second round PCR were 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, they were both identified by PCR. 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, which were identified by enzyme cutting and sequencing.
3. 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. The green fluorescence was obvious under the fluorescent microscope. The different concentration of glycoprotein was added into the transgenic cells and the known chemopreventive agents PDTC and lentinan were as controls. The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dose-dependency with the different concentrations of glycoprotein in a certain range while the controls have not.
This study primary constructed transgenic cell model which can induce the phaseⅡenzyme. It was not only provides a basis for the screening of natural or synthetic cancer chemopreventive agents, but also provides experimental evidence for the Chlorella pyrenoidosa developed as chemopreventive agents.
引文
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