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浙江大学学报（医学版） 2009年第3期38卷 260-264页

作者：沈其陈枢青浙江大学药学院药理毒理与生化药学研究所浙江杭州310058

目的:纯化HSA/IL1ra融合蛋白并对其生物学活性进行检测。方法:采用亲和层析及离子交换层析分离纯化HSA/IL1ra融合蛋白,检测其对IL1引起的对A375 S2的杀伤保护作用。结果:获得的HSA/IL1ra融合蛋白HPLC纯度在98%以上,并且具有与天然IL1ra... 详细信息

目的:纯化HSA/IL1ra融合蛋白并对其生物学活性进行检测。方法:采用亲和层析及离子交换层析分离纯化HSA/IL1ra融合蛋白,检测其对IL1引起的对A375 S2的杀伤保护作用。结果:获得的HSA/IL1ra融合蛋白HPLC纯度在98%以上,并且具有与天然IL1ra相似的、对IL1杀伤A375 S2细胞的保护作用。结论:获得高纯度的HSA/IL1ra融合蛋白并且具有相应的生物学活性。

关键词：受体,白细胞介素1/拮抗剂和抑制剂血清白蛋白重组融合蛋白质类/分离和提纯重组融合蛋白质类/生物合成黑色素瘤

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应用原核细胞表达法体外制备基因重组胰岛素降解酶的活性

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中国临床康复 2005年第31期9卷 83-85页

作者：高琳琳李尧华吴燕川于顺陈彪首都医科大学宣武医院北京老年医学研究所神经生物研究室北京市100053 吉林大学第一临床医院内分泌科

目的:利用原核细胞表达法体外制备基因重组胰岛素降解酶,并分析其活性。方法:实验于2004-05/11在北京老年医学研究所神经生物研究室完成。将编码大鼠胰岛素降解酶的cDNA插入质粒pGEX-4T-1并转化大肠杆菌BL21,使其表达谷胱甘肽巯基转移... 详细信息

目的:利用原核细胞表达法体外制备基因重组胰岛素降解酶,并分析其活性。方法:实验于2004-05/11在北京老年医学研究所神经生物研究室完成。将编码大鼠胰岛素降解酶的cDNA插入质粒pGEX-4T-1并转化大肠杆菌BL21,使其表达谷胱甘肽巯基转移酶为标签的融合蛋白质。凝血酶定点分解融合蛋白,用谷胱甘肽-琼脂糖4B亲合层析方法纯化基因重组胰岛素降解酶。重组胰岛素降解酶的活性应用高压液相色谱进行判断。结果:①胰岛素降解酶cDNA编码区片段的亚克隆:重组质粒亚克隆的cDNA片段为3060bp,编码1019个氨基酸。②胰岛素降解酶在原核细胞的表达与纯化:重组质粒pGEX-rIDE在原核细胞中表达为可溶性组分,培养菌液可收获目的基因重组酶约0.5mg/L,纯化的胰岛素降解酶在SDS-PAGE上表现为单一条带,其表观相对分子质量为110000,与野生型酶相同。③基因重组蛋白质对胰岛素的降解作用:牛胰岛素与基因重组蛋白质的洗脱峰分别出现在34.5和38.3min,将纯化的重组蛋白与胰岛素在37℃下共同孵育后,胰岛素含量明显减少。孵育的最初20min质量变化明显,其峰面积值减少了约50%,但其余时间降解不明显。该蛋白质对14-3-3蛋白的两个亚型没有降解作用。结论:基因重组rIDE具有天然酶的活性,但该活性在孵育20min后胰岛素降解延缓,可能是胰岛素的降解产物反馈抑制了rIDE活性,与酶的自身调节有关。本实验制备了具有生物学活性的基因重组型大鼠胰岛素降解酶,为进一步制备抗胰岛素降解酶单克隆抗体以及探讨胰岛素降解酶与2型糖尿病发生之间的关系打下了基础。

关键词：金属蛋白酶类重组融合蛋白质类/生物合成糖尿病,非胰岛素依赖型

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蓖麻毒素A链与绿色荧光蛋白基因的融合表达和活性测定

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浙江大学学报（医学版） 2005年第3期34卷 201-206页

作者：陈欣虹刘琼詹金彪浙江大学医学院生物化学与分子生物学教研室

　目的:表达蓖麻毒素A链(RTA)与绿色荧光蛋白(GFP)的融合蛋白(GFP-RTA),用于蓖麻毒素A链在细胞内的转运机理研究。方法:采用PCR法从重组质粒pKK233.2-RTA中扩增出蓖麻毒素A链基因,然而将其插入真核表达质粒pEGFPC1,构成GFP-RTA融合基因;... 详细信息

　目的:表达蓖麻毒素A链(RTA)与绿色荧光蛋白(GFP)的融合蛋白(GFP-RTA),用于蓖麻毒素A链在细胞内的转运机理研究。方法:采用PCR法从重组质粒pKK233.2-RTA中扩增出蓖麻毒素A链基因,然而将其插入真核表达质粒pEGFPC1,构成GFP-RTA融合基因;GFP-RTA经PCR扩增,与T载体连接,用内切酶从T-GFP-RTA中切下GFP-RTA片段,插入原核表达质粒pET-28a(+);在BL21(DE3)中表达GFP-RTA融合蛋白,通过金属螯合和层析纯化,MTT法检测融合蛋白的细胞毒性。结果:测序发现插入的融合基因全长序列完全正确,SDS-PAGE鉴定表达产物分子量正确;经诱导表达后,菌体产生绿色荧光,MTT法表明该融合蛋白呈现与RTA相似的细胞毒性。结论:从大肠杆菌表达的蓖麻毒素A链与绿色荧光蛋白的融合蛋白,具有蓖麻毒素A链和绿色荧光蛋白的生物学活性,可用于蓖麻毒素A链在细胞内的转运途径研究。

关键词：蓖麻蛋白蓖麻毒素A链发光蛋白质类重组融合蛋白质类/生物合成基因表达大肠杆菌/代谢聚合酶链反应

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重组Klenow-HSA融合蛋白的构建、纯化及其稳定性

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北京大学学报（医学版） 2002年第6期34卷 684-687页

作者：曾令娥黄晶白惠卿范慧黄大无王露北京医学高等专科学校北京101300 北京大学基础医学院免疫学系

目的 :提高Klenow酶的热稳定性和储藏稳定性。方法 :利用PCR的方法扩增DH5α染色体DNA ,构建编码Klenow和人血清白蛋白第三功能区 (HSA D3)的融合蛋白表达质粒 ,并在大肠杆菌中获得高效表达。分别对Klenow酶和Klenow HSA融合蛋白进行纯... 详细信息

目的 :提高Klenow酶的热稳定性和储藏稳定性。方法 :利用PCR的方法扩增DH5α染色体DNA ,构建编码Klenow和人血清白蛋白第三功能区 (HSA D3)的融合蛋白表达质粒 ,并在大肠杆菌中获得高效表达。分别对Klenow酶和Klenow HSA融合蛋白进行纯化 ,并进行活性及稳定性的研究。结果 :发现Klenow HSA融合蛋白在大肠杆菌中呈可溶性表达。体外实验证实这种融合蛋白具有与Klenow聚合酶相同的活性 ,其稳定性明显提高。结论 :融合蛋白确实能增加Klenow酶的热稳定性和储存稳定性。

关键词： DNA聚合酶 I/生物合成人血清白蛋白重组融合蛋白质类/生物合成 DNA聚合酶 I Klenow片段

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热休克蛋白融合蛋白对人树突状细胞分化成熟的刺激作用

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吉林大学学报（医学版） 2004年第6期30卷 835-838,874页

作者：万敏张培因王宣军吴秀丽卫红飞王燕媚于永利王丽颖邓平张慧杰吉林大学基础医学院分子生物学教研室吉林长春130021 吉林大学基础医学院免疫学教研室吉林长春130021 吉林省长春市中心血站吉林长春130033

目的 :研究热休克蛋白融合蛋白对人树突状细胞分化成熟的刺激作用。方法 :采用 GM- CSF和 IL- 4将人外周血单核细胞诱导成未成熟树突状细胞 ,然后用热休克蛋白融合蛋白刺激其分化成熟 ,以单核细胞条件培养液 (MCM)和模拟的单核细胞条件... 详细信息

目的 :研究热休克蛋白融合蛋白对人树突状细胞分化成熟的刺激作用。方法 :采用 GM- CSF和 IL- 4将人外周血单核细胞诱导成未成熟树突状细胞 ,然后用热休克蛋白融合蛋白刺激其分化成熟 ,以单核细胞条件培养液 (MCM)和模拟的单核细胞条件培养液 (MCM m imic)为对照。以流式细胞仪检测树突状细胞表面分子的表达情况。结果 :热休克蛋白融合蛋白可以刺激树突状细胞表面表达 CD4 0、CD80、CD86和 HL A- A2分子。结论 :热休克蛋白融合蛋白可诱导人树突状细胞分化成熟。

关键词：树突细胞热休克蛋白质类重组融合蛋白质类/生物合成

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Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-Golgi network of 3T3L1 adipocytes

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第2期275卷 1261-1268页

作者： Watson, RT Pessin, JE Univ Iowa Dept Physiol & Biophys Iowa City IA 52242 USA

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes, Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation, Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN, In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6, Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.

关键词： 3T3细胞脂细胞/代谢脂细胞/超微结构脂肪组织/代谢基因文库高尔基体/代谢高尔基体/超微结构绿色荧光蛋白质类发光蛋白质类/生物合成发光蛋白质类/遗传学膜蛋白质类/遗传学膜蛋白质类/代谢诱变神经组织蛋白质类/遗传学神经组织蛋白质类/代谢 Qa-SNARE蛋白质类重组融合蛋白质类/生物合成序列缺失转染动物小鼠

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The splicing factor U1C represses EWS/FLI-mediated transactivation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第32期275卷 24865-24871页

作者： Knoop, LL Baker, SJ St Jude Childrens Res Hosp Dept Dev Neurobiol Memphis TN 38105 USA Univ Tennessee Dept Pathol Memphis TN 38163 USA

EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations, In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1, EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor, Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.

关键词：氯霉素O-乙酰转移酶/遗传学克隆分子基因文库谷胱甘肽转移酶/遗传学 HL-60细胞 HeLa细胞萤光素酶类/遗传学癌基因蛋白质类融合/遗传学癌基因蛋白质类融合/代谢 RNA聚合酶Ⅱ/代谢重组融合蛋白质类/生物合成核糖核蛋白 U1小核/代谢核糖核蛋白类小核/代谢酵母菌酿酒肉瘤 Ewing/遗传学反式激活(遗传学) 转录因子/遗传学转录因子/代谢易位遗传人类

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Construction and expression of functional multi-domain polypeptides in Escherichia coli:: expression of the Neurospora crassa metallothionein gene

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LETTERS IN APPLIED MICROBIOLOGY 2000年第2期30卷 161-166页

作者： Mauro, JM Pazirandeh, M USN Res Lab Ctr Biomol Sci & Engn Washington DC 20375 USA

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and target-ed to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind Cd-109(2+). The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.

关键词： ATP结合匣式转运子氨基酸序列碱基序列镉/代谢载体蛋白质类/代谢大肠杆菌/化学大肠杆菌/遗传学大肠杆菌/代谢大肠杆菌蛋白质类麦芽糖结合蛋白质类金属硫蛋白/生物合成金属硫蛋白/化学金属硫蛋白/遗传学金属硫蛋白/代谢分子序列数据单糖转运蛋白质类粗糙链孢霉/遗传学粗糙链孢霉/代谢寡肽类/生物合成寡肽类/代谢质粒/遗传学蛋白质工程重组融合蛋白质类/生物合成串联重复序列/遗传学

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Expression of β-amyloid precursor protein-CD3γ chimeras to demonstrate the selective generation of amyloid β_1-40 and amyloid β_1-42 peptides within secretory and endocytic compartments

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第45期274卷 32295-32300页

作者： Soriano, S Chyung, ASC Chen, XH Stokin, GB Lee, VMY Koo, EH Univ Calif San Diego Dept Neurosci 0691 La Jolla CA 92093 USA Univ Penn Sch Med Dept Pathol & Lab Med Philadelphia PA 19104 USA

Amyloid beta-protein (A beta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP), We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3 gamma), CD3 gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3 gamma to create an APP chimera predicted to remain in the ER (gamma APP(ER)). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (gamma APP(LYS)). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (gamma APP(Delta Delta)), This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that A beta(1-40) is generated in the early secretory and endocytic pathways, whereas A beta(1-42) is made mainly in the secretory pathway, More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived A beta forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either A beta(1-40) or A beta(1-42).

关键词：氨基酸序列淀粉样β肽类/生物合成淀粉样β肽类/遗传学 CHO细胞仓鼠亚科胞吞作用分子序列数据肽碎片/生物合成重组融合蛋白质类/生物合成转染动物

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A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第2期275卷 1128-1136页

作者： Fensome, AC Rodrigues-Lima, F Josephs, M Paterson, HF Katan, M Chester Beatty Labs CRC Ctr Cell & Mol Biol London SW3 6JB England

Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.

关键词：脑/酶学细胞系内质网/酶学基因文库同工酶类/拮抗剂和抑制剂同工酶类/遗传学同工酶类/代谢动力学肝/酶学镁/代谢哺乳动物 PC12细胞活性氧/生理学重组融合蛋白质类/生物合成重组蛋白质类/拮抗剂和抑制剂重组蛋白质类/代谢鞘磷脂磷酸二酯酶/拮抗剂和抑制剂鞘磷脂磷酸二酯酶/遗传学鞘磷脂磷酸二酯酶/代谢转染动物小鼠大鼠

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