大肠杆菌表达的单链抗体柱复性的研究

对包含体表达的抗乙肝病毒表面抗原(HBsAg)的单链抗体(ScFv)纯化复性进行了探索.尝试了利用金属螯合亲和层析和凝胶层析柱进行柱上在位复性的可行性. 对包含体表达的ScFv进行透析复性与柱复性,比较其相对复性率及蛋白质回收率,发现柱上复性效果优于传统的透析复性.抗HBsAg ScFv经凝胶色谱Sephacyl S-200柱复性的相对复性率为98%, 蛋白质回收率为81%.由于将纯化复性同步进行,简化了操作程序,提高了产品的回收率.     

蛇毒纤溶酶Fibrolase的凝胶过滤色谱复性研究

目的：研究蛇毒纤溶酶Fibrolase的最佳复性方法。方法：使用透析和凝胶色谱法对Fibrolase复性进行研究,比较2种复性方法的相对复性率及蛋白质回收率。结果：2种复性方法的复性率分别为20％、25％,蛋白质回收率分别为16％、5％。结论：凝胶过滤色谱复性优于透析法复性,凝胶过滤色谱复性使蛋白质的复性与纯化同步进行,简化了操作程序,提高了产品回收率。     

大肠杆菌表达的单链抗体柱复性的研究

对包含体表达的抗乙肝病毒表面抗原（HBsAg）的单链抗体（ScFv）纯化复性进行了探索.尝试了利用金属螯合亲和层析和凝胶层析柱进行柱上在位复性的可行性. 对包含体表达的ScFv进行透析复性与柱复性,比较其相对复性率及蛋白质回收率,发现柱上复性效果优于传统的透析复性.抗HBsAg ScFv经凝胶色谱Sephacyl S-200柱复性的相对复性率为98%, 蛋白质回收率为81%.由于将纯化复性同步进行,简化了操作程序,提高了产品的回收率.     

抗SARS 人源单链抗体H12的表达及复性

从SARS免疫抗体库获得的一株抗SARS-CoV人源单链抗体H12,亟待鉴定.为了快速制备大量具有生物活性的单链抗体H12,构建了pET28a-H12原核高表达载体,表达量占菌体总蛋白质30%以上.采用稀释复性和分子筛柱复性两种方法对包涵体蛋白进行复性与纯化,结果显示两种方法都能使得单链抗体复性.与稀释复性法相比,柱复性效果更好,其抗原结合活性是稀释复性法的1.51倍.柱复性后的单链抗体亲和力测定的解离常数Kd为73.5nmol/mL.为进一步研究单链抗体H12的功能奠定了基础.     

抗胰岛素样生长因子1受体单链抗体复性研究

[目的]建立抗人胰岛素样生长因子1受体单链抗体包涵体复性方法。[方法]首先,在96孔板上进行稀释复性,从72种复性液中筛选最佳条件。每孔复性液2 m L,滴入起始浓度1. 5 mg/m L的包涵体溶解液100μL过夜复性。然后,选择最佳复性液与变性液混合在Superdex 75柱上形成1 cm柱长下降5%的变性液梯度,样品按5%柱体积上样进行柱上复性。[结果]最适稀释复性液为C8(50 mmol/L Tris-Cl,GSH/GSSG=5/0. 5 mmol/L,0. 4 mol/L精氨酸,p H 9.0),对应复性率约为78%;以该复性液为基础通过柱上复性,目标蛋白复性率提高到95%,复性样品抗原结合活性良好。[结论]建立了目标蛋白包涵体柱上复性方法,复性率达到95%,产量达到384 mg/L。     

pBV220/hIL-4优化表达及其包涵体复性研究

旨在对人白细胞介素-4(hIL-4)上、下游序列进行优化设计,提高在原核系统中的表达量,并对表达的包涵体进行复性研究提高复性率.利用BioSun的RNA二级结构预测模块辅助设计hIL-4起始密码子(AuG)上、下游的序列,使局部二级结构的自由能满足高表达要求;根据pBV220栽体和原核系统的特点优化引物序列提高hIL-4原核表达量.优化hIL-4包涵体复性条件和方法,提高复性率和生物活性.结果显示,成功构建了pBV220/hIL-4高效表达栽体,原核表达的重组人IL-4占细茵总蛋白的35%以上,明显高于优化前表达量;经过复性研究hIL-4包涵体复性率可达到15%以上,生物活性鉴定发现,纯化的hhIL-4蛋白的比活等同或高于国外同类产品.影响原核表达的条件较多,对表达序列的优化设计可以大大提高蛋白的表达量.针对人IL-4基因序列的优化设计提高了人IL-4基因的表达,复性方法的改良提高了复性率和生物活性.     

蛋白质层析柱复性及工艺评价

蛋白质复性工艺的研究一直是重组蛋白药物研发领域的热点。稀释复性法和透析复性法的蛋白损失较大、复性得率不理想,而层析柱可以完成复性同时纯化,并能在高蛋白浓度条件下得到较高的复性率,有利于规模放大,是近年来最受关注的复性工艺。就层析柱复性工艺进行了归纳,包括凝胶过滤层析、离子交换层析、亲和层析、疏水相互作用层析的复性,并对其复性原理及各自的优缺点进行了比较分析,最后从复性工艺的有效性、优越性以及对于规模化生产的适用性三个角度论述复性工艺的评价方法。     

甲胎蛋白的原核表达及复性优化

构建甲胎蛋白的原核表达载体p ET32a-AFP,对包涵体形式表达的甲胎蛋白进行复性优化。将构建的重组质粒p ET32a-AFP转化入E.coli,IPTG诱导表达后,经亲和层析纯化获得AFP包涵体,通过对复性过程、p H、添加剂等的研究摸索,获得最佳复性条件。当采用添加0.5 mol/L L-精氨酸的一步法透析复性方法,且透析液p H值为8.5,重组人AFP包涵体蛋白起始浓度为1.0 mg/m L时,复性效率最高。该复性方法获得蛋白质具有较高的回收率且操作简便。     

重组包涵体蛋白质的折叠复性

综述了减少包涵体形成、包涵体分离和溶解以及包涵体折叠复性的策略及其最新进展 .详细讨论了包涵体蛋白质折叠复性的基本原则、包涵体折叠复性促进剂和包涵体折叠复性方法     

抗肿瘤血管抗体AA98功能片段VH/κ的脲浓度梯度柱复性

利用稀释法、凝胶过滤、脲浓度梯度凝胶过滤3种方法研究了抗肿瘤血管抗体功能片段VH/κ的体外复性.通过考察复性液中还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)比例、精氨酸浓度、pH值、洗脱速度、变性液中蛋白质浓度、凝胶过滤脲梯度长度等因素,发展了脲梯度凝胶过滤柱复性VH/κ的方法.结果表明,与传统的稀释法和柱复性法相比,脲梯度法复性获得的VH/κ的活性回收率和相对亲和力均有显著提高.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Neuroprotection of striatal neurons against kainate excitotoxicity by neurotrophins and GDNF family members

Neurotrophic factors are regarded as potential therapeutic tools in neurodegenerative disorders. Here, we analysed the protective effects of brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor and neurturin against the excitotoxic damage induced by kainate in striatal neurons in vitro and in vivo. Our results show that the decrease in the number of cultured striatal calbindin-positive neurons induced by kainate was prevented by treatment with any of these factors. To characterize their protective effects in vivo, cell lines overexpressing brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor or neurturin were grafted into the striatum. We found that the numbers of striatal projection neurons (calbindin-positive) and striatal interneurons (parvalbumin- or choline acetyltransferase-positive) were differentially decreased after kainate lesion. These neurotrophic factors prevented the loss of striatal projection neurons and interneurons with differing efficiency: brain-derived neurotrophic factor was the most efficient, whereas neurturin was the least. Our findings show that brain-derived neurotrophic factor, neurotrophin-3, glial cell line-derived neurotrophic factor and neurturin have specific neuroprotective profiles in striatal neurons and indicate that they are specific modulators of the survival of distinct subsets of striatal neurons in pathophysiological conditions.     

NGF and the local control of nerve terminal growth

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response. 1994 John Wiley & Sons, Inc.     

Effect of Tie-2 conditional deletion of BDNF on atherosclerosis in the ApoE null mutant mouse

The reduced expression (haplodeficiency) of the main brain derived neurotrophic factor receptor, namely TrkB is associated with reduced atherosclerosis, smooth muscle cells accumulation and collagen content in the lesion. These data support the concept that brain derived neurotrophic factor of vascular origin may contribute to atherosclerosis. However, to date, no experimental approach was possible to investigate this issue due to the lethality of brain derived neurotrophic factor null mice. To overcome these limitations, we generated a mouse model with a conditional deletion of brain derived neurotrophic factor in endothelial cells (Tie-2 Cre recombinase) on an atherosclerotic prone background (apolipoprotein E knock out) and investigated the effect of conditional brain derived neurotrophic factor deficiency on atherosclerosis. Despite brain derived neurotrophic factor reduction in the vascular wall, mice with conditional deletion of brain derived neurotrophic factor did not develop larger atherosclerotic lesion compared to controls. Smooth muscle cell content as well as the distribution of total and fibrillar collagen was similar in the atherosclerotic lesions from mice with brain derived neurotrophic factor conditional deficiency compared to controls. Finally an extended gene expression analysis failed to identify pro-atherogenic gene expression patterns among the animal with brain derived neurotrophic factor deficiency. In spite of the reduced brain derived neurotrophic factor expression, similar atherosclerosis development was observed in the brain derived neurotrophic factor conditional deficient mouse compared to controls. These pieces of evidence indicate that endothelial derived-brain derived neurotrophic factor is not a pro-atherogenic factor and would rather suggest to investigate the role of other TrkB activators on atherosclerosis.     

Masseteric Nerve Injury Increases Expression of Brain-Derived Neurotrophic Factor in Microglia Within the Rat Mesencephalic Trigeminal Tract Nucleus

The distribution of brain-derived neurotrophic factor was examined in the rat mesencephalic trigeminal tract nucleus after transection and crush of the masseteric nerve. In the intact mesencephalic trigeminal tract nucleus, brain-derived neurotrophic factor was detected in small cells with fine processes. These cells and processes were occasionally located adjacent to tyrosine kinase B receptor-immunoreactive sensory neurons. The transection and crush of the masseteric nerve increased expression of brain-derived neurotrophic factor in the nucleus. The number and size of brain-derived neurotrophic factor-immunoreactive cells and processes were dramatically elevated by the nerve injury. As a result, the density of brain-derived neurotrophic factor-immunoreactive profiles in the mesencephalic trigeminal tract nucleus at 7 days after the injury was significantly higher compared with the intact nucleus. Double immunofluorescence method also revealed that brain-derived neurotrophic factor-immunoreactive cells were mostly immunoreactive for OX-42 but not glial fibrillary acidic protein. In addition, the retrograde tracing method demonstrated that brain-derived neurotrophic factor-immunoreactive cells and processes surrounded retrogradely labeled neurons which showed tyrosine kinase B receptor-immunoreactivity. These findings indicate that the nerve injury increases expression of brain-derived neurotrophic factor in microglia within the mesencephalic trigeminal tract nucleus. The glial neurotrophic factor may be associated with axonal regeneration of the injured primary proprioceptor in the trigeminal nervous system.     

Brain-derived neurotrophic factor levels in the nervous system of wild-type and neurotrophin gene mutant mice

Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.     

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on the ciliary neurotrophic factor receptor alpha chain

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.     

The effect of gp130 stimulation on glutamate-induced excitotoxicity in primary hippocampal neurons

Primary hippocampal neurons from newborn rats treated with glutamate showed clear excitotoxicity. This excitotoxicity could be reversed by treatment of the cells with cytokines of the interleukin-6 family. Stimulation of gp130 on hippocampal neurons resulted in tyrosine phosphorylation of STAT3 and activation of p42 and p44 MAP kinases. Receptors for the interleukin-6 type cytokines are active in membrane bound and soluble form. To address the question whether the neurotrophic effect of interleukin-6 type cytokines requires soluble cytokine receptors we used fusion proteins of interleukin-6 coupled to the soluble interleukin-6 receptor and ciliary neurotrophic factor coupled to the soluble ciliary neurotrophic factor receptor. Ciliary neurotrophic factor was as active as the cytokine-receptor fusion protein, indicating that hippocampal neurons express ciliary neurotrophic factor receptor on the cell surface. In contrast, interleukin-6 was only active at very high concentrations whereas the fusion protein of interleukin-6 coupled to the soluble interleukin-6 receptor (Hyper-IL-6) exhibited high neurotrophic activity at the same concentrations as ciliary neurotrophic factor. These data indicate that interleukin-6 receptor expression is very low on hippocampal neurons and that gp130 stimulation can be used to rescue hippocampal neurons from excitotoxicity.     

Brain-derived neurotrophic factor (BDNF) mediates bone morphogenetic protein-2 (BMP-2) effects on cultured striatal neurones

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein-2 (BMP-2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin-positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP-2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP-2 had a cooperative effect with low doses of brain-derived neurotrophic factor or neurotrophin-3 (but not with other neurotrophic factors such as glial cell line-derived neurotrophic factor, neurturin or transforming growth factor-beta 2) on the number of calbindin-positive striatal neurones. Moreover, BMP-2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP-2 neurotrophic effects. The addition of TrkB-IgG or antibodies against brain-derived neurotrophic factor abolished the effects of BMP-2 on the number and degree of differentiation of calbindin-positive striatal neurones. Indeed, BMP-2 treatment increased brain-derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain-derived neurotrophic factor mediates the effects of BMP-2 on striatal neurones.