利用荧光蛋白对大肠杆菌蛋白质Tat转运系统的研究

探讨了荧光蛋白作为报告蛋白用于蛋白质转运系统研究的可行性 ,结果表明海葵红色荧光蛋白聚集在细胞质内 ,不能转运至周质空间。而水母绿色荧光蛋白在Tat信号肽和Tat转运酶的共同作用下 ,以折叠形式转运至周质空间。通过荧光定量分析表明信号肽保守序列中的双精氨酸是保证绿色荧光蛋白转运及转运效率所必需的 ,且第二个精氨酸比第一个精氨酸更为重要。同时 ,揭示了Tat信号肽需要一定的高级结构才能行使功能 ;Tat信号肽不仅引导蛋白质的转运 ,而且也参与蛋白质的折叠。因此 ,绿色荧光蛋白是非常理想的报告蛋白 ,可用于研究Tat系统 ,但是海葵红色荧光蛋白易于聚集而不适合于此目的。     

Intestinal Translocation of Clinical Isolates of Vancomycin-Resistant Enterococcus faecalis and ESBL-Producing Escherichia coli in a Rat Model of Bacterial Colonization and Liver Ischemia/Reperfusion Injury

The objectives of this study were to develop a rat model of gastrointestinal colonization with vancomycin-resistant Enterococcus faecalis (VRE) and extended-spectrum beta-lactamase (ESBL)-producing E. coli and to evaluate intestinal translocation to blood and tissues after total and partial hepatic ischemia. Methods - We developed a model of rat colonization with VRE and ESBL-E coli. Then we studied four groups of colonized rats: Group I (with hepatic pedicle occlusion causing complete liver ischemia and intestinal stasis); Group II (with partial liver ischemia without intestinal stasis); Group III (surgical manipulation without hepatic ischemia or intestinal stasis); Group IV (anesthetized without surgical manipulation). After sacrifice, portal and systemic blood, large intestine, small intestine, spleen, liver, lungs, and cervical and mesenteric lymph nodes were cultured. Endotoxin concentrations in portal and systemic blood were determined. Results – The best inocula were: VRE: 2.4×10¹⁰ cfu and ESBL-E. coli: 1.12×10¹⁰ cfu. The best results occurred 24 hours after inoculation and antibiotic doses of 750 µg/mL of water for vancomycin and 2.1 mg/mL for ceftriaxone. There was a significantly higher proportion of positive cultures for ESBL-E. coli in the lungs in Groups I, II and III when compared with Group IV (67%; 60%; 75% and 13%, respectively; p:0.04). VRE growth was more frequent in mesenteric lymph nodes for Groups I (67%) and III (38%) than for Groups II (13%) and IV (none) (p:0.002). LPS was significantly higher in systemic blood of Group I (9.761±13.804 EU/mL−p:0.01). No differences for endotoxin occurred in portal blood. Conclusion –We developed a model of rats colonized with resistant bacteria useful to study intestinal translocation. Translocation occurred in surgical procedures with and without hepatic ischemia-reperfusion and probably occurred via the bloodstream. Translocation was probably lymphatic in the ischemia-reperfusion groups. Systemic blood endotoxin levels were higher in the group with complete hepatic ischemia.     

大肠杆菌双顺反子表达载体中绿色荧光蛋白表达水平的研究

构建了一系列含有编码硫氧还蛋白(Trx)和绿色荧光蛋白(GFP)基因的双顺反子重组表达质粒,重点考察双顺反子间紧密相连和重迭的间隔序列.诱导表达后,对表达的GFP进行荧光检测,利用GFP的荧光强度来反映蛋白质的表达水平.结果表明各表达载体所表达的GFP的平均荧光强度差距很大,表明双顺反子间的间隔序列对报告基因表达水平有一定的影响,为实现不同功能基因表达的精确调控奠定了基础.     

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     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

小鼠3T3-L1前脂肪细胞系的增强绿色荧光蛋白标记

细胞模型是研究细胞分化原理以及进行高通量筛选的有效工具。为了建立特异性标记的脂肪细胞分化模型,构建了包括脂肪细胞分化特异性表达基因PPARγ2的启动子在内的载体（pPPARγ2-promoter-EGFP）,用电穿孔方法转染小鼠3T3L1 前脂肪细胞,用显微荧光观察和RT-PCR确认PPARγ2基因的内源表达。结果显示,EGFP基因成功转入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PPARγ2基因的内源性表达。建立了由脂肪组织特异表达基因PPARγ2的表达控制的EGFP标记的小鼠3T3-L1前脂肪细胞系,目前国内外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。     

绿色荧光蛋白标记检测鸦胆子素D抗TMV活性

以绿色荧光蛋白（green fluorescent protein, GFP）为报告基因,将含TMV表达载体的质粒p35S-30B：GFP转化农杆菌EHA 105,通过渗透法把经MMA诱导后的农杆菌悬浮液注射到本氏烟叶片内,测定了鸦胆子素D （Bruceine D） 对烟草植株内TMV的增殖和运动的抑制作用;通过PEG介导法把p35S-30B：GFP转化到本氏烟叶肉细胞原生质体内,测定了Bruceine D对烟草原生质体中TMV增殖的抑制效果.结果表明,在10 μg/mL浓度下,Bruceine D不仅可抑制烟草叶肉细胞原生质体中TMV的增殖,还可以抑制烟草接种叶中TMV向茎部及植株上部叶片移动,且对寄主植物不造成明显的毒害.     

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 .     

Cyclophosphamide-Induced Bacterial Translocation in Escherichia coli C25-Monoassociated Specific Pathogen-Free Mice

The kinetics of bacterial translocation (BTL) from the intestine to the mesenteric lymph nodes (MLN) and the number of peripheral white blood cells (WBC), macrophages in Peyer's patches (PP) and M-cells on the surface of cecal PP after cyclophosphamide (CY) injection were examined in penicillin-G and streptomycin sulfate decontaminated and Escherichia coli C25-monoassociated specific pathogen-free mice. WBC were counted to confirm the immunological state of the mice. Until 8 days after CY injection, the number of WBC, bacteria in MLN and macrophages in PP decreased, but then significantly increased on day 14. The levels again decreased to the control levels on day 16. Although the number of M-cells decreased up to day 8, it did not return to the control level on day 16. These results indicate that BTL is stimulated in an immunopotentiated state after CY injection, and this phenomenon may be closely related to the number of macrophages in the blood and PP.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

3个不同品系小鼠ES细胞的绿色荧光蛋白标记

用电穿孔法将线性化的质粒pEGFP—N3分别导入来自129／ter、C57BL/6J和BALB／c3个品系的小鼠胚胎干细胞：MESPU—13、MESPU—35和MESPU—62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85％的被EGFP稳定标记的细胞株5个(129／ter2个、C57BL/6J1个、BALB／c2个),分别命名为MESPU—13／G1和MESPU—13／G2、MESPU—35／G1、MESPU—62／G1和MESPU—62／G2。从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4群基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征。     

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfp uv ) from Escherichia coli

Background  

Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfp _uv ) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfp _uv from the over-expressing cells.     

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.     

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.     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

Translocation of alpha-synuclein expressed in Escherichia coli

alpha-Synuclein is a major component of Lewy bodies in Parkinson's disease. Although no signal sequence is apparent, alpha-synuclein expressed in Escherichia coli is mostly located in the periplasm. The possibilities that alpha-synuclein translocated into the periplasm across the inner membrane by the SecA or the Tat targeting route identified in bacteria and that alpha-synuclein was released through MscL were excluded. The signal recognition particle-dependent pathway is involved in the translocation of alpha-synuclein. The C-terminal 99-to-140 portion of the alpha-synuclein molecule plays a signal-like role for its translocation into the periplasm, cooperating with the central 61-to-95 section. The N-terminal 1-to-60 region is not required for this translocation.     

绿色荧光蛋白在生命科学研究中的应用

近年来,随着水母Ａｅｑｕｏｒｅａｖｉｃｔｏｒｉａ来源的绿色荧光蛋白（Ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ,ＧＦＰ）在各种异源细胞,如细菌、霉菌、线虫、酵母、果蝇、昆虫细胞、哺乳动物细胞及植物细胞中的表达,ＧＦＰ作为一种新型的报告物在生物学界...