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81.
82.
在分析鸭瘟病毒gB蛋白抗原性的基础上,设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因.将克隆的基因定向插入pET-32a的EcoR Ⅰ和HindⅢ之间,构建了gB蛋白主要抗原域原核表达载体pET-gB1.将pET-gB1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了DPV gB蛋白主要抗原域的高效表达.免疫印迹试验表明获得的表达产物具有良好的反应原性.应用His·Bind亲和层析柱纯化重组DPV gB蛋白,以纯化的重组gB1蛋白作为检测抗原,初步建立了检测鸭瘟病毒抗体的igB1-ELISA.结果表明,抗原的最佳包被浓度为6.5μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490>0.4,且待检血清OD490/阴性血清OD490>2.应用igB1-ELISA对鸭血清样品进行检测,结果表明igB1-ELISA与全病毒包被的iDPV-ELISA符合率达到95.6%. 相似文献
83.
正常小鼠高频心电图时域值和功率谱的研究 总被引:4,自引:1,他引:4
本文用南京新博公司生产的NHE-1000型心电高频信息检测分析仪研究了正常小鼠(昆明种)高频心电图(HF-ECG)的时域值和QRS波群的功率谱。主要结果如下(以正导为例,-X±SD):心率603±88次/min(n=74);P-R间期相对较长。为34.9±4.7ms(n=58),占心动周期的34.9±4.9%,这与人类有很大的不同;QRS波宽9.2±1.2ms,占心动周期的9.2±1.4%(n=74),这一结果与以前的文献报道相差较大。T波宽10.3±3.2ms,占心动周期的10.3±3.2%;Q-T间期19.4±3.2ms,占心动周期的19.5±3.6%;QRS波群峰-峰值(Vp-p)为1.456±0.480mV;T波高0.336±0.115mV;73只动物Ⅱ导联高频切迹总数只有3个,扭挫26个。Ⅱ导联QRS波群的功率谱特点:0—80Hz的相对能量为45.48±15.32%;80—200Hz为43.97±9.95%;200—300Hz为8.89±7.38%;300—1000Hz为1.66±2.74%。 相似文献
84.
85.
Zhou Y Drummond DC Zou H Hayes ME Adams GP Kirpotin DB Marks JD 《Journal of molecular biology》2007,371(4):934-947
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting. 相似文献
86.
87.
Sara Kanje Raminta Venskutonytė Julia Scheffel Johan Nilvebrant Karin Lindkvist-Petersson Sophia Hober 《Journal of molecular biology》2018,430(18):3427-3438
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification. 相似文献
88.
Bradley HJ Liu X Collins V Owide J Goli GR Smith M Surprenant A White SJ Jiang LH 《FEBS letters》2010,584(23):4740-4744
We investigated membrane targeting of the P2X7 receptor (P2X7R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X7R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.
Structured summary
MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416) 相似文献89.
Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains. 相似文献
90.
Recent research has highlighted roles for non-coding RNA i7n the regulation of stress tolerance in bats. In this study, we propose that microRNA could also play an important role in neuronal maintenance during hibernation. To explore this possibility, RT-PCR was employed to investigate the expression of eleven microRNAs from the brain tissue of euthermic control and torpid bats. Results show that eight microRNAs (miR-21, -29b, -103, -107, -124a, -132, -183 and -501) increased (1.2–1.9 fold) in torpid bats, while the protein expression of Dicer, a microRNA processing enzyme, did not significantly change during torpor. Bioinformatic analysis of the differentially expressed microRNA suggests that these microRNAs are mainly involved in two processes: (1) focal adhesion and (2) axon guidance. To determine the extent of microRNA sequence conservation in the bat, we successfully identified bat microRNA from sequence alignments against known mouse (Mus musculus) microRNA. We successfully identified 206 conserved pre-microRNA sequences, leading to the identification of 344 conserved mature microRNA sequences. Sequence homology of the identified sequences was found to be 94.76 ± 3.95% and 98.87 ± 2.24% for both pre- and mature microRNAs, respectively. Results suggest that brain function related to the differentiation of neurons and adaptive neuroprotection may be under microRNA control during bat hibernation. 相似文献