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1.
以融合蛋白形式在大肠杆菌中表达人MCP—1 总被引:2,自引:0,他引:2
将编码人单核细胞趋化蛋白-1(MCP-1)的基因亚克隆到大肠杆菌表达载体pEX31A中,在大肠杆菌中表达出MS2/MCP-1融合蛋白,该表达产物约占菌体总蛋白的15%左右,Western blot检测表明,表达产物可与MCP-1抗体特异反应,采用琼脂糖平板法进行活性测定表明,表达产物具有明显的单核细胞趋化活性,说明N端融合一段细菌蛋白对MCP-1有无趋化活性可能没有影响。 相似文献
2.
叶棋浓 《国外医学:分子生物学分册》1994,16(3):101-105
人单核细胞趋化蛋白-1为一由76个氨基酸组成的单肽链,它既具有特异的单核细胞趋化活性,也具有激活单核细胞的活性,在机体防御,炎症恢复和抗肿瘤等方面起着重要作用。本就MCP-1近几年来的研究进展作一综述。 相似文献
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利用PCR技术和DNA体外重组方法,把作为导向效应细胞到靶部位的单核细胞趋化激活因子(MCAF)和粒细胞巨噬细胞集落刺激因子(GM-CSF)进行基因融合,置于pBV220载体的λPRPL串联启动子下游,构建了SD序列与ATG之间含有不同核苷酸组成的重组质粒pMG01、pMG02和pMG03。pMG01、pMG02和pMG03的翻译起始区都不存在稳定的二级结构,但DH5α(pMG02、DH5α(pMG03)的表达水平远远高于DH5α(pMG01),DH5α(PMG01)几乎没有表达。表达产物经Westernblot检测表明,它能分别与MCAF和GM-CSF抗体发生特异反应。生物学活性测定表明,表达产物具有明显的单核细胞趋化活性和维持hGM-CSF依赖的TF1细胞生长的特性,说明MCAF和GM-CSF的生物学功能是相容的. 相似文献
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以双顺反子表达载体,在大肠杆菌中经IPTG诱导表达了人骨形成蛋白-3羧基端肽段(hBMP-3C),表达量占菌体总蛋白量的18.5%.目的蛋白为25kD、含hBMP-3C端215个氨基酸残基组成的肽段,包括hBMP-3成熟肽和一部分前肽.表达产物以包涵体的形式存在,用含TritonX-100的洗涤液和5mol/L以下脲溶液连续洗涤,可获得较高纯度的重组人骨形成蛋白-3C端肽.经复性处理成可溶性蛋白,植入小鼠肌肉内,第14d组织切片显示有软骨细胞和软骨基质形成,第21d可见成骨细胞和骨基质形成.将rhBMP-3C与脱矿去免疫原性异种骨粒复合后作小鼠肌肉植入试验,21d组织切片上可见硬质骨形成.结果表明:大肠杆菌表达的hBMP-3C经复性后具有诱骨活性,糖基化并非BMP-3活性所必需. 相似文献
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利用人工全合成人胰岛素样生长因子工(hIGF-I)基因,构建了分别以β-半乳糖苷醇(β-gal)的三种即含590、310、280个氨基酸序列片段和一种以ProteinA的B、C结构域(PABC)为载体蛋白的融合型表达质粒,并在大肠杆菌中高效表达了以上各种hIGF-I的融合蛋白产物。通过对羟胺裂解前后的hIGF-I产物进行放射免疫结合测定分析,结果显示以β-Gal690、310、280为载体蛋白,均严重影响与其融合的hIGF-I的免疫原性结构形成,但在PABC-hIGF-I融合蛋白中,载体蛋白PABC无明显的影响作用。这表明在高融合表达低分子量蛋白或肽段中,需选择适宜的载体蛋白,以利于目的产物的功能性结构形成。 相似文献
6.
重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)和人单核细胞趋化激活因子(MCAF)融合蛋白经SephadexG-75和CM-SepharoseFF两步柱层析,获得了电泳纯的GM-CSF/MCAF融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。DotELISA和Westernblot试验表明,该抗血清效价高、特异性好,可分别与GM-CSF/MCAF、GM-CSF和MCAF发生反应。 相似文献
7.
重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)和人单核细胞趋化激活因子(MCAF)融合蛋白经SephadexG-75和CM-SepharoseFF两步柱层析,获得了电泳纯的GM-CSF/MCAF融合蛋白。为进一步研究其结构与功能,我们以纯化的该融合蛋白为抗原免疫家兔制备抗血清。DotELISA和Westernblot试验表明,该抗血清效价高、特异性好,可分别与GM-CSF/MCAF、GM-CSF和MCAF发生反应。 相似文献
8.
rhGM—CSF/LIF融合蛋白基因的克隆及表达 总被引:2,自引:0,他引:2
利用基因重组技术,人工构建了一个编码五肽G-S-G-G-S的基因接头,将GM-CSF和LIF的cDNA相连而构成融合基因,将融合基因载入原核表达载体pBV220后转化大肠杆菌,经热诱导后进行Western印迹反应鉴定证实获得rhGM-CSF/LIF融合蛋白(简称rhgM-LIF)活性测定表明重组的融合蛋白具有两因子双重活性。 相似文献
9.
霍乱毒素B亚基(CTB)是良好的免疫佐剂和载体蛋白。本研究通过定点突变,在CTB基因(ctxB)3′端终止密码前引入了限制性内切酶EcoRI,构建了质粒pMC05。pMC05中CTB与下游lacZ′基因阅读框架相同,转化大肠杆菌后能够表达CTB与β-半乳糖苷酶α肽的融合蛋白;所表达的融合蛋白能与GM1结合,说明融合蛋白保持CTB的基本高级结构和生物学活性;融合蛋白能与抗-CTB抗体结合,说明融合蛋白具有CTB的抗原性。以上结果表明:通过将外源抗原决定簇基因融合至ctxB的3′端,在大肠杆菌中表达融合蛋白,构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响,构建了高效表达融合蛋白的载体-宿主系统。 相似文献
10.
目的:克隆人趋化因子MIP3α,进行原核表达并初步鉴定其趋化活性。方法:从人扁桃体中提取总RNA,进行RTPCR,扩增MIP3α成熟蛋白基因,重组于pET32a(+)载体,转化大肠杆菌BL21TrxB(DE3),进行融合表达,Westernblot验证融合蛋白,金属离子亲和层析,肠激酶酶切,弱阳离子交换层析,得到纯化的MIP3α蛋白,趋化试验鉴定其趋化活性。结果:成功构建了MIP3α天然蛋白的硫氧还蛋白融合表达载体,表达并纯化出MIP3α蛋白,Westernblot证明融合蛋白能与羊抗人MIP3α抗体结合,纯化的MIP3α蛋白能趋化HEK293CCR6稳定转染细胞。结论:构建的天然MIP3α融合表达载体以可溶性蛋白的方式稳定表达MIP3α,初步纯化得到的MIP3α具有趋化HEK293CCR6稳定转染细胞的活性。 相似文献
11.
Expression of monocyte chemoattractant protein-1 by nonenzymatically glycated albumin (Amadori adducts) in vascular smooth muscle cells 总被引:5,自引:0,他引:5
Ichiki T Funakoshi Y Ito K Takeshita A 《Biochemical and biophysical research communications》2000,269(3):666-670
The biological effects of Amadori adducts that are early nonenzymatically glycated protein on vascular cells were poorly defined. We examined the effect of glycated serum albumin (GA) on the expression of monocyte chemoattractant protein-1(MCP-1) that is an important chemokine recruiting monocyte to blood vessel. GA increased MCP-1 mRNA expression with a peak after 3 h of stimulation. The induction of MCP-1 by GA was dose-dependent. The MCP-1 mRNA expression by GA was completely inhibited by PD98059 and genistein that inhibit mitogen activated protein (MAP) kinase kinase and tyrosine kinase, respectively. N-Acetylcysteine, a potent antioxidant, also suppressed the GA-induced MCP-1 expression. These results suggest that GA induces production of reactive oxygen species and activates tyrosine kinase and MAP kinase in VSMC. Activation of these signals results in MCP-1 expression. GA-induced MCP-1 expression may be one of the mechanisms by which the diabetic patients suffer from accelerated atherosclerosis. 相似文献
12.
Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells 总被引:8,自引:0,他引:8
Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo. 相似文献
13.
Monocyte chemotactic protein (MCP-1) is a specific monocyte chemoattractant and activating factor produced by both immune cells (mononuclear phagocytes and lymphocytes) and non-immune cells (parenchymal and stromal cells). In order to define the conditions under which human monocytes express MCP-1, monocytes were exposed to IFN-gamma, IL- lbeta, TNF-alpha, IL-4 or PHA under serum free conditions. There was no significant MCP-1 production by monocytes following exposure to IL-lbeta, TNF-alpha or IL-4. In contrast, stimulation with IFN-gamma resulted in a dose dependent increase in MCP-1 protein and mRNA expression. Simultaneous stimulation with IFN-gamma and IL-1beta or TNF-alpha resulted in no further increase in MCP-1 production. It is concluded that IFN-gamma, primarily a product of T(H)1 T lymphocytes, stimulates the expression of MCP-1 by monocytes. 相似文献
14.
Zhen-Bo Liu 《Biochemical and biophysical research communications》2009,386(4):703-708
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Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment 总被引:7,自引:0,他引:7
Fillion I Ouellet N Simard M Bergeron Y Sato S Bergeron MG 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(12):7353-7361
Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model. 相似文献
17.
Zhao Z McCloud B Fleming R Klempner MS 《Biochemical and biophysical research communications》2007,358(2):528-533
Matrix metalloproteinase 9 (MMP-9) is selectively upregulated in erythema migrans (EM) lesions with acute Lyme disease. This study explored whether upregulation of MMP-9 was associated with monocyte chemoattractant protein 1 (MCP-1) production, and Borrelia burgdorferi (B. burgdorferi) could induce MCP-1 production in vivo and in vitro. The results indicated that expression of MCP-1 was significantly increased in U937 cells by B. burgdorferi. The activity of MMP-9 could be elevated by recombinant MCP-1 (rMCP-1) in U937 cells. MMP-9 was not upregulated by B. burgdorferi in fibroblasts. However, the expression of MCP-1 was significantly increased in the presence of B. burgdorferi in fibroblasts. The level of MCP-1 in EM lesions and in serum of patients with acute Lyme disease was also significantly elevated compared to that for healthy controls. The secreted MCP-1 may affect the production of MMP-9 in fibroblasts and/or macrophages. 相似文献
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Wang G Siow YL O K 《American journal of physiology. Heart and circulatory physiology》2001,280(6):H2840-H2847
Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation. 相似文献
20.
Wen Y Gu J Vandenhoff GE Liu X Nadler JL 《American journal of physiology. Heart and circulatory physiology》2008,294(4):H1933-H1938
Monocyte chemoattractant protein (MCP)-1 plays a key role in atherosclerosis and inflammation associated with visceral adiposity by inducing mononuclear cell migration. Evidence shows that mouse peritoneal macrophages (MPM) express a 12-lipoxygenase (12/15-LO) that has been clearly linked to accelerated atherosclerosis in mouse models and increased monocyte endothelial interactions in both rodent and human cells. However, the role of 12/15-LO products in regulating MCP-1 expression in macrophages has not been clarified. In this study, we tested the role of 12/15-LO products using MPM and the mouse macrophage cell line, J774A.1 cells. We found that 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] increased MCP-1 mRNA and protein expression in J774A.1 cells and MPM. In contrast, 12(R)-HETE, a lipid not derived from 12/15-LO, did not affect MCP-1 expression. 15(S)-HETE also increased MCP-1 mRNA expression, but the effect was less compared with 12(S)-HETE. MCP-1 mRNA expression was upregulated in a macrophage cell line stably overexpressing 12/15-LO (Plox-86 cells) and in MPM isolated from a 12/15-LO transgenic mouse. In addition, the expression of MCP-1 was downregulated in MPM isolated from 12/15-LO knockout mice. 12(S)-HETE-induced MCP-1 mRNA expression was attenuated by specific inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein kinase (p38). 12(S)-HETE also directly activated NADPH oxidase activity. Two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium chloride, blocked 12(S)-HETE-induced MCP-1 mRNA. Apocynin attenuated 12(S)-HETE-induced MCP-1 protein secretion. These data show that 12(S)-HETE increases MCP-1 expression by inducing PKC, p38, and NADPH oxidase activity. These results suggest a potentially important mechanism linking 12/15-LO activation to MCP-1 expression that induces inflammatory cell infiltration. 相似文献