Use of Green Fluorescent Protein (GFP) and Its Homologs for in vivo Protein Motility Studies

Green fluorescent protein (GFP) and its homologs are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells. In particular, based on GFP and GFP-like proteins a number of techniques have been developed that can be used either to estimate protein mobility in living cells, or to introduce a distinctive fluorescent signal in order to track the movement of labeled molecules directly. Considerable progress in the development of such technologies in the last two or three years motivates us to reevaluate the present scope of biotechnological instruments in studies of protein movement in cells.     

增强型绿色荧光蛋白的真核表达和纯化

根据编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的开放读码框(open reading frame,ORF)设计引物,PCR方法扩增出5'端带His标签的EGF PORF,利用杆状病毒表达系统构建表达EGFP基因的重组杆状病毒DNA分子,转染sf9细胞.取细胞...     

绿色荧光蛋白与 HCV 核心蛋白的融合及在大肠杆菌中的高效表达

利用基因工程重组技术获得了绿色荧光蛋白（ｇｆｐ）基因与ＨＣＶ核心蛋白基因的嵌合体,并在大肠杆菌中高效表达了４８ｋＤａ的融合蛋白,经Ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ免疫活性分析证实,融合蛋白仍具有ｃｏｒｅ抗原的三个免疫活性部位,同时用荧光显微镜观察并用荧光光度计测定了大肠直菌表达的融合蛋白的荧光光谱,结果证实,我们在大肠杆菌中表达的ＧＦＰ－ｃｏｒｅ融合蛋白既能发射易于检测的绿色荧光,又具     

Kinetic Analysis of Maturation and Denaturation of DsRed, a Coral-Derived Red Fluorescent Protein

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.     

小鼠3T3-L1前脂肪细胞系的特异性标记(英文)

用增强绿色荧光蛋白特异性标记小鼠 3T3 L1前脂肪细胞系 .构建paP2 promoter EGFP载体 ,电穿孔转染小鼠 3T3 L1前脂肪细胞 ,显微荧光观察和RT PCR确认aP2基因的内源表达 .EGFP基因转入 3T3 L1前脂肪细胞 ,观察到细胞分化过程中EGFP表达和脂肪积累 .RT PCR分析表明 ,EGFP代表了稳定而真实的aP2基因的内源性表达 .建立了由脂肪组织特异表达基因aP2的表达控制的EGFP标记的小鼠 3T3 L1前脂肪细胞系 ,目前尚未见用同样方法对前脂肪细胞进行特异性标记 .该细胞系将为脂肪细胞分化机理研究以及为抗肥胖症和抗糖尿病药物筛选提供有力工具 .     

绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡     

绿色荧光蛋白表达载体pXS75－GFP的构建及在嗜热四膜虫中的应用

为获得能够用于构建嗜热四膜虫蛋白定位的载体,该研究将GFP基因与镉（Cd2＋）诱导的四膜虫金属硫蛋白基因（MTTl）启动子序列和终止子序列融合,获得表达载体pXS75-GFP。通过同源重组和抗性筛选,pXS75-GFP载体携带的目的基因整合入四膜虫MTTl位点,在cd2＋诱导下实现GFP融合蛋白的可控表达。将α－tubulin基因ATUl克隆JN－pXS75－GFP中,重组质粒pXS75－GFP－ATUl通过基因枪转化入四膜虫细胞,在巴龙霉素筛选下获得稳定的α-tubulin－GFP过表达细胞株。激光共聚焦显微镜观察α－tubulin．GFP的定位,结果显示,α－tubulin—GFP融合蛋白在四膜虫细胞中表达并分布于皮层上,表明pXS75．GFP载体可用于嗜热四膜虫功能蛋白的定位分析。     

芜菁原生质体的分离及绿色荧光蛋白的瞬时表达

目的：分离芜菁叶片原生质体,建立蛋白质在芜菁原生质体的瞬时表达系统。方法：以津田芜菁成叶为试材,酶解分离原生质体;通过PEG介导的转化,将编码绿色荧光蛋白（GFP）的瞬时表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中GFP的表达情况。结果：分离出大量的津田芜菁原生质体,并获得了较高的转化效率,GFP在整个原生质体中都有表达。结论：建立了津田芜菁原生质体瞬时表达系统。     

Construction of a New Bacterial Cloning Vector Using a Mutant Green Fluorescent Protein as an Indicator

A new bacterial cloning vector, pGreenLD, derived from the triple substitution mutated Aequorea v/ctor/a green fluorescent protein(GFP-S65A, V68L, S72A), when expressed in E. coli produced colonies which showed yellow-green colour under daylight and strong green fluorescence under long-wave ultraviolet light. It can be a useful vector for selecting foreign DNA fragment which was inserted into multiple cloning site based on the loss of the yellow-green color/green fluorescence of E. coli cells attributable to the insertional inactivation of GFP production.     

绿色荧光蛋白及其在GMOs生态监测中的应用

绿色荧光蛋白（green fluorescent protein,GFP）是在海洋无脊椎动物水母（Aequorea victoria）中获得的一种由238个氨基酸组成的多肽。该多肽通过翻译后加工形成生色基团,产生稳定的荧光,而且这种荧光很容易被检测。GFP作为动、植物以及微生物基因工程研究上的一种广泛的选择标记,具有检测灵敏度高、操作简便、不需要添加任何底物或辅助因子等优点,更重要的是利用GFP可对GMOs进行快速、原位、实时、活体监测。本文概括介绍了GFP的特性、改造及其检测,并从生态学角度论述了GFP在GMOs生态监测研究中的应用及其发展前景。     

High-Resolution Crystal Structure and Redox Properties of Chloroplastic Triosephosphate Isomerase from Chlamydomonas reinhardtii

Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.     

Expression of Green Fluorescent Protein from Bacterial and Plastid Promoters in Tobacco Chloroplasts

The expression of the green fluorescent protein reporter gene (gfp) from the bacterial trc and plastid rrn and psbA promoters has been compared in transplastomic tobacco plants produced by microprojectile bombardment. The homoplasmic nature of the regenerated plants was confirmed by Southern blot analysis. Northern blot analysis indicated that plants expressing gfp from the rrn promoter contained 3-fold more gfp RNA than plants containing the psbA promoter and 12-fold more than plants with the trc promoter. Immunoblot analysis and fluorescence spectroscopy indicated that plants expressing gfp from the rrn promoter contained approximately 90-fold more green fluorescent protein (GFP) than plants containing the psbA or trc promoters. This study demonstrates that the bacterial trc promoter is significantly weaker than the plastid rrn promoter for expression of gfp in tobacco chloroplasts.     

绿色荧光蛋白基因导入胡萝卜愈伤组织并获得表达

目的:将绿色荧光蛋白基因(green fluorescent protein,GFP)重组到胡萝卜愈伤组织细胞中,使其获得表达,为今后利用GFP基因作为植物报告基因提供条件。方法:通过冻融法将含有GFP基因的重组表达载体PBI1121转入到根癌农杆菌EHA105中,再利用根癌农杆菌介导的方法将GFP基因导入到胡萝卜愈伤组织细胞中,经过除菌和抗性筛选后观测转化结果。结果:荧光显微镜观测到被转化的愈伤组织在受蓝光激发后发出绿色荧光,利用PCR法扩增出约740bp的目的基因片断。结论:GFP基因在胡萝卜愈伤组织细胞中获得了表达。     

用绿色荧光蛋白研究军团菌与寄主的相互关系

原生动物作为军团菌的天然寄主,在军团菌的生存、增殖、毒力和抗逆性等方面起着重要的作用。通过多次转化筛选,获得了一种高量表达绿色荧光蛋白基因gfpmut2的自发突变质粒。该质粒在嗜肺军团菌细胞内稳定复制和表达;转化了该质粒的嗜肺军团菌在自然光下即可发出明亮的绿色荧光。以转化菌饲喂嗜热四膜虫BF1株后,在荧光显微镜下能清楚观察到细菌在细胞内的形态变化、增殖和裂解宿主细胞的过程。为研究嗜肺军团菌与原生动物寄主的相互关系提供了一种简单而直观的方法。     

A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.     

表达绿色荧光蛋白突变体的重组伪狂犬病毒的构建及其增殖特性

将绿色荧光蛋白突变体M 1(EGFPS14 7/P)基因融合到伪狂犬病毒 (PRV)非必需糖蛋白 gG的第 8个氨基酸下游 ,通过同源重组、空斑纯化和PCR筛选获得能表达M 1并导致gG基因部分缺失的重组病毒 gG-/M1 。重组病毒经Southern杂交、Western印迹和荧光观察证实构建正确。纯化的重组病毒以低感染指数接种PK 15细胞 ,在感染早期 (6h)就能观察到荧光 ,随着病毒的增殖 ,荧光逐渐增强 (2 4～ 36h) ,直至完全病变 ,荧光淬灭。进一步对重组病毒gG-/M1 与亲本株gG-/LacZ 、野毒株的增殖特性进行比较 ,发现 3种毒株在增殖滴度上无显著差异。上述结果表明构建的PRVgG-/M1 突变株能作为活细胞示踪实时监测病毒感染的动态分析。     

乙型肝炎病毒e抗原基因与绿色荧光蛋白基因的融合及其表达

质粒pS65T含有T7启动子驱动的绿色荧光蛋白突变型gfpS65T基因,经修饰后,在其中插入乙型肝炎病毒e抗原（HBeAg）基因,使两基因同框成为融合基因。在大肠杆菌BL21中由于T7启动子的控制,高效表达了具有双功能（抗原性和发光性）的融合蛋白（GFP－HBeAg）。融合蛋白MW为52kDa,N端有6个组氨酸残基,因而用Ni金属螯合层析柱对融合蛋白进行了分离纯化,利用诊断HBV的ELISA试剂盒检测了融合蛋白的抗原性,在荧光显微镜下观察到了融合蛋白的绿色荧光。并探讨了用其组装成新型免疫诊断试剂的可能性。     

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

抗菌肽CM4与绿色荧光蛋白融合基因的真核载体构建及表达

目的：构建绿色荧光蛋白（GFP）与抗菌肽CM4融合基因的真核表达载体。方法：采用递归PCR（rPCR）将GFP基因与CM4基因通过一段核苷酸片段连接成GFP-CM4融合基因,构建真核表达载体pcDNA3-GFP-CM4,用脂质体介导转染人K562白血病细胞,用MTT检测其活性。结果：融合基因可在K562细胞中表达,抗菌肽CM4的表达可抑制K562细胞的生长。结论：为全面了解抗菌肽的生物学活性及其在基因治疗中的可能作用提供了重要的理论依据。     

大鼠谷氨酰胺转运蛋白SNAT2-EGFP融合蛋白的表达与鉴定

谷氨酰胺转运蛋白是中枢神经系统中一种重要的中性氨基酸转运蛋白,对谷氨酰胺的跨膜转运十分重要。为了更方便地研究大鼠谷氨酰胺转运蛋白2(SNAT2)在细胞膜上的表达与定位,利用亚克隆技术将增强型绿色荧光蛋白(EGFP)构建于SNAT2的C端,通过菌液PCR、酶切和DNA测序鉴定重组真核表达质粒;将测序正确的重组质粒瞬时转染人胚胎肾细胞(HEK293T cells),用Western blot和激光共聚焦电子显微镜荧光检测技术鉴定SNAT2-EGFP的表达与亚细胞定位。结果表明,SNAT2-EGFP融合蛋白重组质粒在细胞中表达并正确定位于细胞膜上。SNAT2-EGFP融合蛋白重组质粒的成功构建为今后深入研究SNAT2的结构和功能提供了一个有效的工具。