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以2008年诺贝尔化学奖为背景的命题
例1 水母体内有两种发光蛋白,一种是水母素,它在有钙离子的环境中就能发出蓝光;另一种是绿色荧光蛋白,它吸收了水母素发出的蓝光后。发出波长较长的绿色荧光。科学家可以对多种基因进行荧光标记,根据活体内的荧光颜色变化,判断生物活动进程。荧光标记不仅可以标记蛋白在哪里,还能标记活体内的生物事件。 相似文献
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《东南大学学报》2015,(3)
为制备可视化的转移消失蛋白(MIM)的I-BAR结构域重组体,克隆了顺联绿色荧光蛋白(GFP)探针编码序列的MIM-I-BAR基因.在6xHis标签原核表达质粒上成功构建了DNA序列.同时,实现了未标记荧光探针基因的MIM-I-BAR质粒的构建以作实验对照.成功转染至BL21(DE3)大肠杆菌细胞后,GFP偶联的MIM-I-BAR(MIM-I-BAR-GFP)蛋白表现出很强的可视荧光,该表达产物可方便的通过目测、荧光显微镜、免疫印迹和紫外可见分光光度计等多种手段进行检测.此外,在考察不同条件下的蛋白表达效率过程中发现,带有GFP探针的MIM-I-BAR重组蛋白在温度为10℃时产率最高,而并非37℃.这一特征与非荧光标记的MIM-I-BAR明显不同.研究证实该最佳表达温度条件适用于重组蛋白产品中量制备.所开发的带有荧光探针的MIM-I-BAR蛋白产品及其制备工艺在科学研究、生物医学应用以及药物开发过程中均有较高的应用价值. 相似文献
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目的：探讨增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）在中华仓鼠卵巢细胞（CHO-DHFR）细胞中的作用。方法：将增强型绿色荧光蛋白基因的真核表达载体pcDNA3．1（＋）-EGFP，转染至培养的中华仓鼠卵巢细胞（Chinese Hamster Ovary, CHO-DHFR^- ）中。结果：成功表达并产生绿色荧光。结论：证明EGFP是一种良好的报告基因和筛选标志．为进一步研究应用最广泛的哺乳动物细胞表达系统一CHO细胞表达系统奠定了基础。 相似文献
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《实验室研究与探索》2020,(2)
绿色荧光蛋白(GFP)在生物体内具有发射高效的荧光特性,故可作为无生物毒性的标记蛋白而被广泛应用于分子生物学、细胞生物学以及药学等领域,其发现和应用是科学研究领域的重要里程碑。GFP带来的技术革命主要源自于其内部发色团的神奇特性,因此研究发色团的激发态性质具有重要意义。该实验将科研成果转换为实验教学内容,设计了"绿色荧光蛋白发色团激发态动力学行为的研究性实验"。实验内容包括文献调研、模型构建、电子结构计算与动力学模拟以及数据处理与分析4部分。通过该教学实践,学生的实验方案设计能力、计算过程排错能力以及实验结果分析能力都得到了显著提高。使学生在科研论文的写作和大学生创新项目的申请中取得了突出的成绩。 相似文献
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以2008年诺贝尔化学奖为背景的命题例1水母体内有两种发光蛋白,一种是水母素,它在有钙离子的环境中就能发出蓝光;另一种是绿色荧光蛋白,它吸收了水母素发出的蓝光后,发出波长较长的绿色荧光。科学家可 相似文献
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Fu Y Wang SQ Liu YP Wang GP Wang JT Gong SS 《Journal of Zhejiang University. Science. B》2008,9(4):299-305
Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies. 相似文献
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INTRODUCTION Because of its low molecular weight and ability to fluoresce independently (George, 1997), the new molecular tag, green fluorescent protein (GFP), has become more and more popular after Prasher et al.(1992) cloned its cDNA in 1992. There are many reports describing the co-expression of GFP and a specific antibody or cytokine gene, with the fusion protein expressing the fluorescent activity and bio-logical activity of the complement protein (Haraguchi et al., 1999; Mclean… 相似文献
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INTRODUCTION Soil-borne pathogens, including Pythium spp. and Fusarium spp., cause significant yield losses in horticulture and agriculture crops (Mao et al., 1997). Current practices for controlling plant diseases are based largely on disease resistant crops, cultivation management in fields and application of synthetic pesticides (Elizabeth and Emmert, 1999). Biological control using antagonistic microbes to reduce the use of chemical pesticides in a system of integrated plantdisease … 相似文献
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Wang JY Wu XY Zhang Z Du XF Chai RY Liu XH Mao XQ Qiu HP Wang YL Lin FC Sun GC 《Journal of Zhejiang University. Science. B》2008,9(10):802-810
The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTSI, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS 1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTSI machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into △mgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTSI and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes. 相似文献
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Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins 总被引：2，自引：0，他引：2
Yan XB Chen Z Luo DH Xu XY Wu W Zhou LF 《Journal of Zhejiang University. Science. B》2005,6(4):295-300
Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed,which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191:1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT＞BNT＞C191). To the different fragment COREs of BT,N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa＞1-126 aa＞1-58aa＞ 127-172 aa＞59-126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis. 相似文献
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Zhang Xin Zhang Bing-xin Zhang Zhen Shen Wei-feng Yang Ching-hong Yu Jing-quan Zhao Yu-hua 《Journal of Zhejiang University. Science. B》2005,6(8):770-777
Fusarium head blight (FHB) caused byFusarium graminearum is a devastating disease that results in extensive yield losses to wheat and barley. A green fluorescent protein (GFP) expressing
plasmid pRP22-GFP was constructed for monitoring the colonization of two biocontrol agents,Brevibacillus brevis ZJY-116, on the spikes of barley and their effect on suppression of FHB. Survival and colonization of theBrevibacillus brevis ZJY-1 andBacillus subtilis ZJY-116 strains on spikes of barley were observed by tracking the bacterial transformants with GFP expression. Our field
study revealed that plasmid pRP22-GFP was stably maintained in the bacterial strains without selective pressure. The retrieved
GFP-tagged strains showed that the bacterial population fluctuation accorded with that of the rain events. Furthermore, both
biocontrol strains gave significant protection against FHB on spikes of barley in fields. The greater suppression of barley
FHB disease was resulted from the treatment of barley spikes with biocontrol agents before inoculation withF. graminearum.
Project supported by the National Natural Science Foundation of China (No. 30230250) and Science and Technology Committee
of Zhejiang Province (No. 2003C22029), China 相似文献
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Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant. 相似文献
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柴明良 汪炳良 KIM Jae-yeoul LEE Jong-min KIM Doo-hwan 《Journal of Zhejiang University. Science. B》2003,(3)
INTRODUCTIONCreepingbentgrass (AgrostispalustrisHuds.)isanoutstandingcool seasonspeciesavailableforuseongolfcourseputtinggreens,tees,andcloselymowedfairways.Althoughco lonialbentgrass (AgrostistenuisSibth .Fl.Ox en .)isnotwelladaptedtotheverylowmowingheig… 相似文献
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增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),也称周期蛋白或DNA聚合酶的辅助蛋白,是真核细胞合成所必需的核蛋白,在DNA复制中起重要作用。前期实验发现日本七鳃鳗肝脏cDNA文库的表达序列标签(Expressed Sequence Tag,EST)中存在与高等脊椎动物pcna基因同源的序列。提取日本七鳃鳗(Lampetra japonica)肝脏组织RNA,通过RT-PCR方法扩增七鳃鳗pcna基因,对其进行生物信息学分析,并将Lj-pcna基因成功构建到pGFP-N2真核表达载体上,重组质粒PGFP-N2-Lj-pcna转染人Hela细胞,荧光显微镜下观察有荧光蛋白的表达。日本七鳃鳗pcna基因的真核表达载体成功构建和转染,为探讨七鳃鳗pcna基因功能研究及其它七鳃鳗相关研究提供条件。 相似文献