重组人源脑红蛋白的高效表达、纯化及鉴定

脑红蛋白(Neuroglobin,NGB)是新发现的主要表达于脊椎动物神经元及视网膜中的第三类携氧珠蛋白。已有诸多报道认为脑红蛋白在缺氧缺血性脑损伤的神经保护过程中,作为活性氧簇(ROS)的清除剂或缺氧信号的感受器起重要作用,但其神经保护作用的具体机制不明。显然,实现该蛋白的制备对于研究其功能具有重要作用。基于此,通过RT-PCR从人胎脑中扩增出脑红蛋白cDNA并克隆到原核表达载体pBV220,通过测序鉴定正确后转化至大肠杆菌HB101中诱导表达,表达产物超声破碎后经凝胶过滤柱Sephacryl S-200和阴离子交换柱Q Sepharose FF纯化,最后通过Sephadex G-25脱盐。经15% SDS-PAGE和Western blot检测及质谱和蛋白序列分析鉴定制备的蛋白样品为脑红蛋白。本文采用两步法实现了脑红蛋白高效特异性纯化,为后期基于脑红蛋白神经保护作用的功能及机制研究奠定了重要基础。     

脑红蛋白ELISA检测试剂盒的研发与应用

目的:利用ELISA技术研发一种灵敏、快速检测人血清中脑红蛋白(Ngb)含量的检测试剂盒,并探讨其在正常人血清样本检测中的应用。方法:采用热诱导的原核表达体系获得重组人源脑红蛋白(rhNgb),通过凝胶过滤和阴离子交换层析等制备rhNgb纯品;将适量rhNgb纯品免疫动物获得抗rhNgb单克隆抗体和多克隆抗体;用双抗体夹心ELISA技术制备rhNgb-ELISA检测试剂盒;用该试剂盒对410例正常人血清中Ngb的含量进行检测,其中包括1.5岁以下婴儿血清55例、19～70岁成人血清355例。结果:用制备的rhNgb-ELISA试剂盒检测发现Ngb在正常人血清中的分布与年龄存在明显的相关性,表现为"两头高、中间低"的趋势,婴儿和老年人血清中Ngb的含量分别为1.6708±0.4945和2.1962±0.6703μmol/L,而中年人血清中Ngb的含量为0.3622±0.0716μmol/L。结论:采用双抗夹心ELISA技术并结合严格的质量控制,研发了高效灵敏的rhNgb-ELISA检测试剂盒,并初步获得了正常人血清中Ngb的含量分布情况,为临床缺血及缺氧性脑损伤等相关疾病的辅助诊断治疗提供了参考信息。     

戊二醛聚合对猪、牛和人血红蛋白免疫学特性的影响

猪、牛、人血红蛋白或其相应的戊二醛聚合体分别经皮下或腹腔多次免疫小鼠,ELISA检测抗血清IgG滴度;免疫印迹法分别检测小鼠抗猪、牛、人血红蛋白或其相应的戊二醛聚合体IgG与猪、牛、人血红蛋白或其相应的戊二醛聚合体之间的交叉反应,探讨戊二醛聚合对不同种类血红蛋白免疫学特性的影响.结果表明:猪、牛、人血红蛋白的免疫原性均...     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Immunogenicity of Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

In the preceding paper (Protein J. 25, pages 37–49, 2005), we reported the preparation and oxygen-binding properties of peptides that form stable complexes with heme mimic. The design of the peptides was based on the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). In the present work, the heme-peptides were each administered into mice, either as emulsions in adjuvant (both for injections and boosters) or intravenously as solutions in phosphate-buffered saline. Antibody (Ab) responses, monitored up to 14 weeks after the first administration, showed that when the heme-peptides were injected with adjuvant they stimulated Ab responses against the immunizing peptide, which in most cases bound to the correlate protein (Mb or Hb). However these heme-peptides were non-immunogenic when administered in PBS intravenously. It is concluded that heme-peptides:(a) would not trigger an adverse immune response if used for transfusion purposes.     

Characterization of Klebsiella Isolates from Natural Receiving Waters and Comparison with Human Isolates

Two hundred sixty-six strains of Klebsiella pneumoniae isolated from natural water sources in geographically diverse areas (Florida, Massachusetts, and Oregon) were analyzed to determine the serotype, biochemical, virulence, and antimicrobial susceptibility differences between these natural strains and human Klebsiella isolates. Sixty of 72 defined serotypes were found among 210 typable strains. Geographic patterns were present, but in general were not pronounced among serotypes. Reactions with 28 biochemical tests showed percentage responses which were very similar to the summaries of primarily human Klebsiella isolates (as reported by Edwards and Ewing, 1972) and that represented diverse geographic sampling. Virulence studies in representative strains showed no geographic variability and little difference from comparable hospital patient-obtained isolates. In contrast to human hospital isolates, strains demonstrated 90% or greater susceptibility to all antibiotics except ampicillin and carbenicillin; and in further contrast, there was little multiple antibiotic resistance beyond that with ampicillin and carbenicillin.     

A Biophysical Model for Integration of Electrical, Osmotic, and pH Regulation in the Human Bronchial Epithelium

A dynamical biophysical model for the functioning of an epithelium is presented. This model integrates the electrical and osmotic behaviors of the epithelium, taking into account intracellular conditions. The specific tissue modeled is the human bronchial epithelium, which is of particular interest, as it is the location of the most common lethal symptoms of cystic fibrosis. The model is implemented in a modular form to facilitate future application of the code to other epithelial tissue by inputting different transporters, channels, and geometric parameters. The model includes pH regulation as an integral component of overall regulation of epithelial function, through the interdependence of pH, bicarbonate concentration, and current. The procedures for specification, the validation of the model, and parametric studies are presented using available experimental data of cultured human bronchial epithelium. Parametric studies are performed to elucidate a), the contribution of basolateral chloride channels to the short-circuit current functional form, and b), the role that regulation of basolateral potassium conductance plays in epithelial function.     

Neuroglobin and cytoglobin. Fresh blood for the vertebrate globin family

Neuroglobin and cytoglobin are two recently discovered members of the vertebrate globin family. Both are intracellular proteins endowed with hexacoordinated heme-Fe atoms, in their ferrous and ferric forms, and display O₂ affinities comparable with that of myoglobin. Neuroglobin, which is predominantly expressed in nerve cells, is thought to protect neurons from hypoxic–ischemic injury. It is of ancient evolutionary origin, and is homologous to nerve globins of invertebrates. Cytoglobin is expressed in many different tissues, although at varying levels. It shares common ancestry with myoglobin, and can be traced to early vertebrate evolution. The physiological roles of neuroglobin and cytoglobin are not completely understood. Although supplying cells with O₂ is the likely function, it is also possible that both globins act as O₂-consuming enzymes or as O₂ sensors. Here, we review what is currently known about neuroglobin and cytoglobin in terms of their function, tissue distribution and relatedness to the well-known hemoglobin and myoglobin. Strikingly, the data reveal that O₂ metabolism in cells is more complicated than was thought before, requiring unexpected O₂-binding proteins with potentially novel functional features.     

Induction of Tolerance to a Recombinant Human Enzyme,Acid Alpha-Glucosidase,in Enzyme Deficient Knockout Mice

When knockout mice are used to test the efficacy of recombinant human proteins, the animals often develop antibodies to the enzyme, precluding long-term pre-clinical studies. This has been a problem with a number of models, for example, the evaluation of gene or enzyme replacement therapies in a knockout model of glycogen storage disease type II (GSDII; Pompe syndrome). In this disease, the lack of acid alpha-glucosidase (GAA) results in lysosomal accumulation of glycogen, particularly in skeletal and cardiac muscle. Here, we report that in a GAA-deficient mouse model of GSDII, low levels of transgene-encoded human GAA expressed in skeletal muscle or liver dramatically blunt or abolish the immune response to human recombinant protein. Of two low expression transgenic lines, only the liver-expressing line exhibited a profound GAA deficiency in skeletal muscle and heart indistinguishable from that in the original knockouts. The study suggests that the induction of tolerance in animal models of protein deficiencies could be achieved by restricting the expression of a gene of interest to a particular, carefully chosen tissue.     

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain.

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.     

Crucial Roles of Glu60 in Human Neuroglobin as a Guanine Nucleotide Dissociation Inhibitor and Neuroprotective Agent

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric G_i/o proteins and inhibits reductions in cAMP concentration, leading to protection against cell death. In the present study, we created human E60Q Ngb mutant and clarified that Glu60 of human Ngb is a crucial residue for its GDI and neuroprotective activities. Moreover, we investigated structural and functional properties of several human Ngb mutants and demonstrated that the neuroprotective effect of human Ngb is due to its GDI activity and not due to its scavenging activity against reactive oxygen species.     

建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

旨在构建AIP1基因CRISPR/Cas9敲除体系,获得高效、稳定、永久性AIP1敲除的人胚肾细胞株(293T)。针对AIP1的外显子设计3个20 bp的sg RNA(sp1、sp2和sp3)。与PX458载体连接,构建PX458-sg RNA敲除表达载体。T7E1实验评估敲除效率。将敲除效率最高的sg RNA与lenti CRISPRv2慢病毒载体连接,构建lenti CRISPRv2-sg RNA敲除表达载体,将阳性重组质粒导入病毒包装细胞293T中,收集病毒上清液转染293T细胞。对敲除成功的293T细胞通过有限稀释法构建敲除AIP1基因的稳定细胞株。Western blot测定转染后293T细胞中AIP1蛋白的表达量。结果显示,测序证实AIP1的3个靶序列分别正确插入PX458表达载体中,T7E1实验证实AIP1sg RNAsp2靶向敲除效率最高;成功构建lenti CRISPRv2-sg RNAsp2 AIP1敲除表达载体,并包装病毒,感染293T细胞;Western blot证实获得稳定的AIP1基因表达缺失的293T细胞株。建立了能稳定敲除AIP1基因的CRISPR/Cas9慢病毒系统,成功获得AIP1敲除的293T稳定细胞株。     

Study of the Adsorption of Human Hemoglobin to Silver (Ag) Nanoparticle Surface for the Detection of the Unfolding of Hemoglobin

In the present report, we focused on the detail study of the optical properties and structural characterization of the Ag NPs for the nanobioconjugate analysis and detection of the conformational structural change of the Hb. The detail optical and structural analysis of Ag NPs has been studied from UV–Vis absorption, emission spectrum, XRD, and HRTEM study. The proteins/Hb are attached immediately onto Ag NPs surface when NPs touch the biological fluids, forming protein corona (PC), which gives their biological identity. The NPs-PC bioconjugate is, more specifically, the true identity of NPs in the physiological world. The adsorption of Hb with Ag NP surfaces has been studied by monitoring the soret band and tryptophan band of Hb. The dynamics of the Hb adsorption on the Ag NPs showed the time constant of surface binding t₁?=?5.79 min and 10.23 min and surface reorganization t₂?=?500 min and 251.75 min with the use of small and large concentrations of Ag NPs, respectively. The absorption peak shape and size around the wavelength, λ ≈ 406.2 nm of the bioconjugate has been examined by Gaussian and Lorentz curve fitting analysis. The bioconjugate along with the PC formation has been analyzed by HRTEM images and DLS observations. The tertiary deformation of Hb and energy transfer efficiency connecting Ag NPs and Hb are discussed from the emission-quenching phenomenon. The change of the secondary structural elements (α-helix, β-sheets, intermolecular aggregates, intramolecular aggregates) of the bioconjugate has been analyzed from FTIR spectrum.

     

Biophysical, Biological, and Cytochemical Features of a Virus Associated with Transplantable Hamster Tumors

A virus resembling type C murine leukemia viruses, which is associated with transplantable hamster tumors, was partially characterized with respect to certain biological, biophysical, and cytochemical features. As determined by electron microscopy, high concentrations of the virus appeared in the blood plasmas of tumor-bearing hamsters. Hamsters inoculated with virus concentrates did not show gross evidence of disease, and preliminary attempts to infect various hamster cells in tissue culture were unsuccessful. A line of cells from a virus-containing tumor which had been established in tissue culture released large numbers of the virus into the supernatant fluids by budding. The buoyant density peak of virus concentrates in potassium tartrate density gradients was 1.13 g/cm(3) by ultraviolet absorption and electron microscopic analysis. Acridine orange staining and nuclease digestion methods have indicated that the virus is probably a ribonucleic acid virus.