绿色荧光蛋白和荧光素酶

绿色荧光蛋白和荧光素酶基因是基因工程中广泛使用的两个报告基因。本文从绿色荧光蛋白和荧光素酶的来源、性质、发光机理、检测方法和用途等方面论述了两者的区别。     

关于绿色荧光蛋白发色团激发态动力学行为的实验设计与实践

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。     

绿色荧光蛋白基因在转基因金鱼中的整合与表达

通过分子克隆技术将鲤鱼β-肌动蛋白启动子(carpβ—actin promoter A)与编码绿色荧光蛋白(GFP)的cD-NA融合构建成能在真核生物体内表达的质粒载体pAGFP。质粒pAGFP经Pvu线性化后采用显微注射法导入金鱼受精卵，在胚胎发育初期，经紫外光激发能观察到少数胚胎发绿色荧光。在通过PCR实验检测出的5个阳性个体染色体DNA中，经点杂交实验证实有2个个体染色体基因组上整合了GFP基因。     

水生植物凤眼莲Ca2＋-ATPase的原核表达与蛋白纯化

从水生植物凤眼莲叶片中提取总 RNA,经 RT-PCR扩增出Ca2＋-ATPase基因片段,经限制性内切酶(Sma I,Not I)酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCR和质粒双酶切鉴定、序列测定确认,证实成功地构建了Ca2＋-ATPase基因融合表达载体,．转化菌经IPTG诱导表达,获得了大小约48 kD的可溶性目的蛋白,与预期相吻合．利用谷胱甘肽琼脂糖凝胶4B(Gluta-thione Sepharose 4B)亲和介质对重组蛋白进行纯化,获得了高纯度的目的蛋白．     

绿色荧光蛋白简介——配合高中化学新课标教材的实施

以高中化学新课标教材中有关氨基酸、蛋白质的知识为切入点,介绍绿色荧光蛋白的结构特点、生色团与发光特性、优点及应用,为一线教师提供前沿化学知识.     

人超氧化物歧化酶（SOD）基因的原核表达与蛋白纯化

从人直肠癌细胞株Colo320中提取总RNA,经RT-PCR扩增出SOD基因片段,经限制性内切酶（BamHI,Sinai）酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCR和质粒双酶切鉴定、序列测定确认,证实成功地构建了人SOD基因融合表达载体．转化菌经IPTG诱导表达,SDS-PAGE显示有与预期大小约44kD相吻合的融合蛋白带,获得了可溶性表达的目的蛋白,采用Glutathione Sepha—rose4B亲和层析对重组蛋白进行纯化,获得了纯度很高的目的蛋白．     

绿色荧光蛋白的发现与发展——2008年诺贝尔化学奖简介

生物发光是一个很普遍的生物学现象.早期的生物研究大多涉及分类、形成和生态方面,后来重点转移到生态、生化的研究,目前已进入分子生物学的研究水平.从不同生物体内提取的荧光蛋白的结构、性质不尽相同.迄今,腔肠动物门多管水母属的维多利亚多管水母(aequorea vietoria)的发光蛋白系统研究得较为深入.     

对虾白斑病毒核糖核苷酸还原酶基因原核表达载体的构建及蛋白表达、纯化

采用PCR方法扩增对虾白斑病毒核糖核苷酸还原酶基因,插入到pGEX-4T-2表达载体上构建出带有目的基因的重组质粒pGEX-4T-2RR,然后将重组质粒转化大肠杆菌.转化菌经IPTG诱导后大量表达重组蛋白,通过降低诱导温度获得可溶性表达的重组蛋白,采用Glutathione Sepharose 4B亲和层析对重组蛋白进行纯化,获得了高纯度的目的蛋白.     

面向应用创新型本科生培养的生物化学实验教学设计 ——以GFP蛋白的表达分离纯化为例

为提高生物化学实验的教学效果,培养学生的创新性、应用性思维,在生物化学实验中以绿色荧光蛋白为对象,设计蛋白质表达与纯化的综合性实验,并以该实验为基础创新了自主实验考核方式.通过实验材料的优化、实验教学内容的改进,提升了该课程的教学质量;以学生自主设计实验并实施的方式对其进行考核,通过考核方式和规则的完善,增强了学生的创...     

少棘蜈蚣候选毒素多肽κ-SLPTX-Ssm1b的原核表达与纯化

κ-SLPTX-Ssm1b是从少棘蜈蚣的c DNA文库中获得的一类功能未知的毒素,它与钾通道抑制剂κ-SLPTX-Ssm1a高度同源,由50个氨基酸残基构成,含有3对二硫键的多肽分子.利用p ET-43-His-SUMO原核表达载体,通过E.coli SHuffleTM菌种自动诱导表达,随后利用镍柱和RP-HPLC纯化,并通过MALDI-TOF质谱鉴定其分子量为5532.1870 Da,与理论值5532.3 Da相符.其产量为1 mg/L,为进一步鉴定其电生理活性奠定了基础.     

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

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…     

激光扫描共聚焦显微镜原位检测核酸及蛋白质的实验教学设计

为实现实验教学与科研工作的紧密结合,该教学设计采用激光扫描共聚焦显微镜在细胞原位检测核酸和绿色荧光蛋白,分析融合蛋白的亚细胞定位。通过2个经典的检测方案,使学生更好地理解和掌握激光扫描共聚焦显微镜的检测原理、操作步骤及结果分析方法。     

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

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.     

Relative bioavailability and pharmacokinetic comparison of two different enteric formulations of omeprazole

In order to comply with the requirements for a drug listed in China, the study was developed to compare the pharmacokinetics and relative bioavailability of two different enteric formulations of omeprazole (OPZ) in healthy Chinese subjects. A total of 32 volunteers participated in the study. Plasma concentrations were analyzed by nonstereospecific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method. After administration of a single 40-mg dose of the two OPZ formulations, the comparative bioavailability was assessed by calculating individual AUC_0−t (the area under the concentration-time curve from time zero to the last measurable concentration), AUC_0−∞ (the area under the concentration-time curve extrapolated to infinity), C _max (the maximum observed concentration), and T _peak (the time to C _max) values of OPZ, 5-hydroxyomeprazole (OH-OPZ), and omeprazole sulfone (OPZ-SFN), respectively. The 90% confidence intervals (CIs) of AUC_0−t , AUC_0−∞, and C _max were 85.4%–99.0%/88.8%–98.6%/87.6%–99.4%, 85.5%–99.2%/89.0%–98.6%/88.5%–101.3%, and 72.3%–87.6%/79.6%–91.1%/88.4%–99.1% for OPZ/OH-OPZ/OPZ-SFN, respectively, and T _peak values did not differ significantly. In this study, the test formulation of OPZ in fasting healthy Chinese male volunteers met the Chinese bioequivalance standard to the reference formulation based on AUC, C _max, and T _peak.     

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass

INTRODUCTIONCreepingbentgrass (AgrostispalustrisHuds.)isanoutstandingcool seasonspeciesavailableforuseongolfcourseputtinggreens,tees,andcloselymowedfairways.Althoughco lonialbentgrass (AgrostistenuisSibth .Fl.Ox en .)isnotwelladaptedtotheverylowmowingheig…     

A study of the relationship between expression level of TRF1 protein and telomerase activity in human acute leukemia

INTRODUCTION Telomeres are distinctive DNA-protein struc-tures that cap the ends of linear chromosomes.It is very important to keep the chromosomes stabilization.Telomerase activity is closely linked to attainment of cellular immortality,a step in carcinogenesis,while lack of such activity contributes to cellular senes-cence.Telomerase is activated in more than85%ofmalignant tumors(Hiayma et al.,1997).Human te-lomeric repeat binding factor1(TRF1)is a telomere associated with proteins a…     

Expression of bone morphogenetic protein 2, 4, and related components of the BMP signaling pathway in the mouse uterus during the estrous cycle

The objective was to investigate the expression of bone morphogenetic protein (BMP) family members in the mouse uterus during the estrous cycle by real-time polymerase chain reaction (PCR) and immunohistochemistry. Uterine samples from Swiss ICR mice were collected and dissected free of surrounding tissue. One uterine horn was snap frozen in liquid nitrogen immediately after collection and stored at −80 °C for RNA extraction, and the other was fixed in 40 mg/ml paraformaldehyde at room temperature for immunolocalization of BMP2 protein. Real-time PCR analysis showed that the expression level of Bmp2 was significantly higher at proestrus than at estrus and metestrus (P<0.05). The relative abundance of Bmp4 exhibited significant fluctuations, but there were no statistically significant differences between the expression levels of Bmp2 and Bmp4 (P>0.05). The expression levels of Bmpr1a and Bmpr2 remained unchanged during estrous cycles. However, the level of Bmpr1b mRNA decreased significantly at estrus (P<0.05), increasing subsequently at metestrus. Furthermore, the level of Bmpr1b mRNA was significantly lower than those of Bmpr1a and Bmpr2 mRNA at the corresponding stages (P<0.05). All three receptor-regulated Smads (R-Smads) detected were differentially expressed in the mouse uterus and the expression levels of Smad1 and Smad5 were significantly higher than that of Smad8 (P<0.05). In addition, the expression level of Smad4 did not change substantially throughout the estrous cycle. Immunohistochemical experiments revealed that BMP2 protein was differentially expressed and localized mainly in the uterine luminal and glandular epithelial cells throughout the estrous cycle. In conclusion, our results provide information about the variation in the mRNA levels of Bmp2 and Bmp4 and related components of the BMP signaling pathway. The data provide quantitative and useful information about the roles of endometrial BMP proposed and demonstrated by others, such as the degradation and remodeling of the endometrium.