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本文对咖啡酸苯乙酯对大肠癌细胞β_catenin蛋白表达的影响进行研究。实验数据表明不同浓度CPAE作用后SW480细胞细胞β_catenin蛋白质含量有不同程度的下降;不同浓度CPAE作用后HCTH16细胞细胞β_catenin蛋白质含量有不同程度的下降。 相似文献
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近年来,基因组测序、编辑与合成技术日新月异,推动了基因型"设计"和"合成"能力的突飞猛进,同时也使人工细胞的表型检测成为合成生物学发展的瓶颈环节之一。对于细胞功能的快速测试与评价,单细胞分析技术具有重要意义与前景,但理想的解决方案需要具备活体无损、非标记式、提供全景式表型、能分辨复杂功能、快速高通量且低成本、能与组学分析联动等特征。以此为出发点,文章重点介绍了基于非标记式分子光谱学的单细胞功能表征、分选与组学技术体系的进展,并讨论了该领域的关键问题与发展方向。多种光谱技术之间扬长避短的运用与多模态成像,结合高通量的光谱激活细胞分选技术及下游单细胞组学技术,正构建与拓展着一条连接光谱学与遗传学的广阔桥梁。这一桥梁不仅为细胞工厂的高通量、全景式表型检测与筛选提供全新的解决方案,还将推动"单细胞精度的光谱表型组-功能基因组"作为一种新的生物大数据类型,服务于"数据科学"驱动下的合成生物技术。 相似文献
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目的应用双向电泳及串联质谱技术,研究不同剂量三氯乙烯(TCE)诱导L-02肝细胞蛋白质表达谱的差异.方法L-02肝细胞分别用不同剂量TCE和溶剂对照(DMSO)处理后,提取细胞总蛋白,双向凝胶电泳分离蛋白质组成分,银染显色,经ImagerMaster 2D Platinum 5.0软件分组分析比较四组图谱,并对差异蛋白斑点进行基质辅助激光解吸/电离飞行时间串联质谱(MALDl-TOF-TOF-MS)分析鉴定.结果得出差异较明显的蛋白质斑点15个,不同剂量TCE刺激后表达分别上调、下调或消失,提示TCE可引起肝细胞蛋白表达发生改变.通过质谱鉴定和lPI human数据库检索,鉴定了其中9个TCE诱导L-02肝细胞相关的蛋白质.结论这些结果为深入研究TCE毒作用相关的蛋白质及TCE毒作用的分子标志物提供依据,并为阐明TCE毒作用的分子机制提供线索. 相似文献
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目的应用双向电泳及串联质谱技术，研究不同剂量三氯乙烯（TCE）诱导L-02肝细胞蛋白质表达谱的差异。方法L-02肝细胞分别用不同剂量TCE和溶剂对照（DMSO）处理后，提取细胞总蛋白，双向凝胶电泳分离蛋白质组成分，银染显色，经ImagerMaster 2D Platinum 5．0软件分组分析比较四组图谱，并对差异蛋白斑点进行基质辅助激光解吸／电离飞行时间串联质谱（MALDI-TOF-TOF-MS）分析鉴定。结果得出差异较明显的蛋白质斑点15个，不同剂量TCE刺激后表达分别上调、下调或消失，提示TCE可引起肝细胞蛋白表达发生改变。通过质谱鉴定和IPI human数据库检索，鉴定了其中9个TCE诱导L-02肝细胞相关的蛋白质。结论这些结果为深入研究TCE毒作用相关的蛋白质及TCE毒作用的分子标志物提供依据，并为阐明TCE毒作用的分子机制提供线索。 相似文献
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组成生物体的蛋白质大多数是在细胞质中的核糖体上合成的,各种蛋白质合成之后要分别运送到细胞中的不同部位,以保证细胞生命活动的正常进行。有的蛋白质要通过内质网膜进入内质网腔内,成为分泌蛋白;有的蛋白质则需穿过各种细胞器的膜,进入细胞器内,构成细胞器蛋白。探索这些跨膜蛋白质如何克服能量上的障碍穿过疏水的脂双层膜是近20年来细胞分子生物学研究的一个热点。德裔美国科学家君特·布洛贝尔就因在70年代中期首先提出蛋白 相似文献
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目的:构建真核表达IL-18-GPI融合基因的表达载体,观察融合蛋白在哺乳动物细胞中的表达动力学及生物学活性,为进一步研究细胞因子作为免疫增强剂在临床肿瘤治疗中的应用奠定基础.方法:通过RT-PCR的方法从脂多糖刺激的外周血淋巴细胞中钓取IL-18基因,通过共用限制性酶切位点与GPI相连形成pcDNA-IL-18-GPI真核表达质粒.将质粒转染CHO细胞后建立稳定表达融合蛋白的细胞株用于融合蛋白的提取.对提取纯化的蛋白进行生物学特性、蛋白质转移分析和γ-IFN诱导实验.结果:获得714 bp的核酸序列并构建重组质粒pcDNA-IL-18-GPI.SDS-PAGE和West-blot显示在CHO细胞中表达的IL-18-GPI融合蛋白分子量约为27.5 kD,该蛋白具有明显的蛋白转移和诱生γ-IFN的作用.结论:IL-18-GPI融合蛋白是一种潜在的肿瘤疫苗增强剂,具有良好的应用前景. 相似文献
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博雅 《大科技．科学之谜》2010,(7):44-45
<正>自从DNA双螺旋结构被发现以来的相当长时间里,科学家认为是DNA中的碱基排列顺序构成了遗传密码,并由它来决定蛋白质的类型,蛋白质决定了生物体的大小、外形和其他许多性状。含有基因的DNA在细胞核内,而蛋白质的合成地点在细胞 相似文献
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Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study, we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) polyethylene glycol 8000 (PEG 8000) and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can, therefore, be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channels. 相似文献
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Crispin Szydzik Khashayar Khoshmanesh Arnan Mitchell Christian Karnutsch 《Biomicrofluidics》2015,9(6)
Microfluidic based blood plasma extraction is a fundamental necessity that will facilitate many future lab-on-a-chip based point-of-care diagnostic systems. However, current approaches for providing this analyte are hampered by the requirement to provide external pumping or dilution of blood, which result in low effective yield, lower concentration of target constituents, and complicated functionality. This paper presents a capillary-driven, dielectrophoresis-enabled microfluidic system capable of separating and extracting cell-free plasma from small amounts of whole human blood. This process takes place directly on-chip, and without the requirement of dilution, thus eliminating the prerequisite of pre-processed blood samples and external liquid handling systems. The microfluidic chip takes advantage of a capillary pump for driving whole blood through the main channel and a cross flow filtration system for extracting plasma from whole blood. This filter is actively unblocked through negative dielectrophoresis forces, dramatically enhancing the volume of extracted plasma. Experiments using whole human blood yield volumes of around 180 nl of cell-free, undiluted plasma. We believe that implementation of various integrated biosensing techniques into this plasma extraction system could enable multiplexed detection of various biomarkers. 相似文献
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Li JF Zhang J Zhang Z Hu YL Zhang SQ 《Indian journal of clinical biochemistry : IJCB》2010,25(3):319-325
Leptin, a 16 kDa nonglycosylated hormone, is produced by mature adipocytes and functions primarily in the hypothalamus to
reduce food intake and body weight. To explore a new approach for high-level expression of human Leptin in Escherichia coli, the human Leptin gene, synthesized according to the published sequence, was cloned into the vector pET32a to construct a
fusion expression plasmid: Trx–Leptin/pET32a. Our data showed that more than 40% of the fusion protein Trx–Leptin was expressed
in soluble form. After purified by Ni-IDA affinity chromatography, cleaved by enterokinase and applied Ni-IDA affinity chromatography
again, purified Leptin with homogeneity over 96% was achieved. The bio-functional experiments of purified Leptin showed a
significant reduction in food intake and body weight of female mice treated with Leptin by comparing with control mice, and
it indicated that the purified Leptin has full biological activity. In addition, our expression system was a very low-cost
and efficient prokaryotic expression system. So taken together, our results demonstrated that our expression system of bio-active
Leptin provided a new method for producing Leptin in big scale and would be widely applied in commercial Leptin producing
industries. 相似文献
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BackgroundHuman is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression.ResultsIn the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions.ConclusionsThis study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris. 相似文献
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Ajaikumar Sukumaran Rhema Elizabeth Thomas R. Arun Krishnan Thushara Thomas Riji Thomas Deepa K. Vijayan Jofy. K. Paul D. M. Vasudevan 《Indian journal of clinical biochemistry : IJCB》2022,37(3):349
Upon SARS CoV-2 infection, humoral immune system triggers production of anti-SARS CoV-2 IgM and IgG antibodies. Currently, antibodies against SARS CoV-2 spike protein receptor binding domain play a central role in disease protection, making them potential target for in vitro diagnostics applications. This study determines the expression level and sustainability of anti-receptor binding domain (RBD) SARS CoV-2 IgG in post COVID-19 patients. Anti-RBD SARS CoV-2 IgG antibodies in patient serum were analysed by standardised indirect ELISA using SARS CoV-2 spike receptor binding domain protein and HRP conjugated anti-human IgG antibody (anti-h IgG). The study was conducted using 35 adult patient samples with confirmed SARS CoV-2 infection. Additionally, correlation between antibody response after each stage and disease symptoms in post COVID-19 patients were studied. Maximum antibody titre was seen at Day 40 and decreased relatively to Day 180 in antibody positive samples when compared with controls. Overall, more IgG antibody expression is observed in patients who suffered from loss of smell and taste at Day 40. 71% of the positive subjects in this study showed high SARS CoV-2 IgG antibody concentration of above 10 ng/mL and 37% showed strong antibody concentration above 20 ng/mL at the peak of seroconversion. 相似文献
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协同理论为新能源装备智造化转型提供新思路。基于新能源装备全生命周期，试突破常规智能制造系统边界，从目标、流程、信息和资源等四个维度分析了协同智造基本活动并识别关键智造技术，将协同智造主体划分至云平台、研发平台、信息平台、制造平台、服务平台和资源平台等六个主要模块，通过关键智造技术嵌入应用构建离散式新能源装备协同智造体系，使智造主体通过各平台实现“四维协同”模式。基于“四维协同”构建新能源装备协同智造体系运行的要素指标体系，运用引入差异化权值的直觉模糊层次分析模型确定要素权重。实证表明，目标协同和信息协同是影响新能源装备协同智造的关键要素。 相似文献
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Yun Suk Huh Chang-Moon Jeong Ho Nam Chang Sang Yup Lee Won Hi Hong Tae Jung Park 《Biomicrofluidics》2010,4(1)
A protein separation technology using the microfluidic device was developed for the more rapid and effective analysis of target protein. This microfluidic separation system was carried out using the aqueous two-phase system (ATPS) and the ionic liquid two-phase system (ILTPS) for purification method of the protein sample, and the three-flow desalting system was used for the removal of salts from the sucrose-rich sample. Partitioning of the protein sample was observed in ATPS or ILTPS with the various pHs. The microdialysis system was applied to remove small molecules, such as sucrose and salts in the microfluidic channel with the different flow rates of buffer phase. A complex purification method, which combines microdialysis and ATPS or ILTPS, was carried out for the effective purification of bacteriorhodopsin (BR) from the purple membrane of Halobacterium salinarium, which was then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and matrix-assisted laser desorption∕ionization time-of-flight. Furthermore, we were able to make a stable three-phase flow controlling the flow rate in the microfluidic channel. Our complex purification methods were successful in purifying and recovering the BR to its required value. 相似文献
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Dengue fever is one of the major health problems in India. Interaction with specific receptor(s) at the cell surface is one
of the first events in the pathogenesis of Dengue virus. However, relatively little is known about these receptors. Cellular
receptors in human monocytes and mouse neural cells are main target for the viral infection. The envelope protein of the virus
(E-protein) plays important role in attachment of virus to target cells and their interaction with cellular receptors. The
modulation of receptor gene(s) and/or protein(s) can be used as a method for interfering with virus entry and can thus become
a new method for disease prevention. The receptors can be purified by affinity chromatography using E-protein as ligand. It
has been reported that addition of highly sulfated heparan sulfate prevents E-protein binding to target cells suggesting that
heparan sulfate is utilized by dengue envelope protein to bind to target cells. 相似文献
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BackgroundThe heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis.ResultsWe produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41°C.ConclusionsAlthough, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore provides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets.How to cite: Harnischfeger J, Beutler M, Salzig D, et al. Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.002 相似文献