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1.
利用大肠杆菌鞭毛展示的随机肽库筛选TNF—α拮抗须 总被引:1,自引:0,他引:1
TNF-α是一种在机体抗感染,抗肿瘤过程中发挥重要作用的细胞因子,其对机体具有保护和损伤两方面的作用,为了探讨新的抑制TNF-α所致炎症损伤等反应的手段,构建了细菌鞭毛递呈的随机肽库,利用构建得到的肽库,进行TNF-α特异性结合肽的筛选工作。经过5轮筛选及DNA测序,共得到6条小肽编码序列。其中2条序列中含有-V--N-WG的相同序列框架。进行6条序列与TNF-α结合力的确证后,选择了其中的4条肽序列进行人工化学合成,纯化及鉴定。利用L929细胞及MTT法对4条小肽进行活性测定,检测其对TNF-α的抑制活性。结果表明,在TNF-α对L929细胞毒性为30%左右,含同源序列框架的2条肽可抑制90%左右的TNF-α活性。 相似文献
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为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础 相似文献
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TNF-α是一种在机体抗感染、抗肿瘤过程中发挥重要作用的细胞因子,其对机体具有保护和损伤两方面的作用,为了探讨新的抑制TNF-α所致炎症损伤等反应的手段,构建了细菌鞭毛递呈的随机肽库,利用构建得到的肽库,进行TNF-α特异性结合肽的筛选工作。经过5轮筛选及DNA测序,共得到6条小肽编码序列。其中2条序列中含有一V-N-WG的相同序列框架。进行6条序列与TNF-α结合力的确证后,选择了其中的4条肽序列进行人工化学合成、纯化及鉴定。利用L929细胞及MTT法对4条小肽进行活性测定,检测其对TNF-α的抑制活性。结果表明,在TNF-α对L929细胞毒性为30%左右时,含同源序列框架的2条肽可抑制90%左右的TNF-α活性。 相似文献
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为了寻找能够模拟胰岛素生物活性的小肽,以胰岛素多克隆抗体为靶标,筛选噬菌体展示随机C7C环肽库.3轮筛选后,通过ELISA方法挑取与靶分子特异性结合的15个阳性克隆,测序获得两条序列,分析所得序列并合成相应短肽.通过细胞生物学活性检测,小肽CPTSQANSC(ZJ1)能够竞争性的抑制胰岛素与其受体的结合,并对正常小鼠和四氧嘧啶诱导的糖尿病小鼠,都有明显的降血糖作用.上述结果表明,小肽CPTSQANSC具有胰岛素样生物学活性.而小肽CVQPSHSSC(ZJ2)表现出胰岛素拮抗活性,能引起正常小鼠血糖升高.这表明筛选到了能够模拟胰岛素表位的短肽CPTSQANSC,可能为治疗胰岛素依赖性糖尿病提供了新线索. 相似文献
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白细胞介素2亲和性配体的筛选 总被引:3,自引:0,他引:3
白细胞介素2(IL-2)及其受体拮抗剂的研究对免疫抑制药物的研制具有重要作用.抗白细胞介素2受体α链中和性单克隆抗体5G1(抗Tac型抗体)能够特异性地阻断IL-2与其受体的结合.因此,5G1可作为目标分子被用来在噬菌体展示肽库中筛选白细胞介素2的亲和性配体.经过4轮亲和性筛选以及5G1亲和活性的测定,6个具有明显5G1亲和活性的噬菌体克隆被发现.DNA序列分析结果显示出,所得到的肽序列具有明显的保守性,即SSFT(L/P)I.该序列与IL-2受体α链没有同源性.因此,SSFT(L/P)I可能模拟了IL-2受体α链上的一个不连续表位(mimotope),为白细胞介素2亲和性配体片段. 相似文献
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目的:评价原核表达、纯化的6×His-硫氧还蛋白(TRX)一人肿瘤坏死因子α(TNFα)抑制肽-C端抗炎酸性尾巴融合蛋白的生物学功能。方法:在大肠杆菌中分别表达带His标签的TRX对照蛋白及TRX蛋白融合的人TNFα抑制肽-抗炎酸性尾巴融合蛋白,并对2种蛋白进行N^2+金属螯合层析纯化,采用MTT法检测纯化后的蛋白及化学合成多肽抑制TNFα标准品对L929细胞的细胞毒活性。结果:与对照蛋白相比,融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成多肽均能拮抗TNFα标准品对L929细胞的细胞毒作用。结论:融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成肽均能有效拮抗TNFα的生物学作用,为今后发展抑制TNFα为主的抗炎生物药物奠定了基础。 相似文献
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应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。 相似文献
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Chemical synthesis of long-chain oligodeoxyribonucleotides was studied by the method of block condensation in a liquid phase. A method for elongating the chain and the synthesis of oligothymidylic acids up to 80-mer are described. 相似文献
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A 13-mer oligoribonucleotide can act as a ribozyme for the specific self-cleavage of a 41-mer oligoribonucleotide substrate in the presence of Mg2+. The two sequences involved correspond to the self-cleavage hammerhead structure of the virusoid of lucerne transient streak virus. The Michaelis-menten kinetic parameters for the reaction were; Km 1.3 microM, Vmax 0.012 microM min-1, kcat 0.5 min-1. The 13-mer RNA is the smallest ribozyme so far reported. A DNA analogue of the 13-mer can not substitute for the RNA in the reaction. 相似文献
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An 11-mer peptide taken from a subsequence of the human protein ubiquitin was synthesized. The peptide has been fully characterized by NMR spectroscopy using chemical shift analysis and by NOE measurements. The conformation was calculated using state of the art MD methods of protein chemistry. A hairpin conformation was found which is to a large part identical with the structure of this peptide fragment within the human ubiquitin. The surprising result that already an 11-mer peptide adopts a hairpin conformation in aqueous solution is discussed in terms of initials sites for protein folding. 相似文献
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C. Schönbach Kiyoshi Miwa Masaaki Ibe Hajime Shiga Kiyoshi Nokihara Masafumi Takiguchi 《Immunogenetics》1996,45(2):121-129
HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell
recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate
the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated
that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers.
These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues.
Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well
as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains
at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects
at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer
residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.
Received: 29 May 1996 / Revised: 5 August 1996 相似文献
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Masaaki Ibe Yuki Ikeda Moore Kiyoshi Miwa Yutaro Kaneko Shumpei Yokota Masafumi Takiguchi 《Immunogenetics》1996,44(4):233-241
The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the
C-terminus were main anchor residues for HLA-A*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus
are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using
a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and
the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there
was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic
hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of
the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction
of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer
peptides to HLA-A*2402 molecules. 相似文献
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Marise Ouellet 《生物化学与生物物理学报:生物膜》2006,1758(9):1235-1244
We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. 31P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, 2H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes. 相似文献
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We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. (31)P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, (2)H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes. 相似文献
20.
Sun Y Li T Chen H Zhang K Zheng K Mu Y Yan G Li W Shen J Luo G 《The Journal of biological chemistry》2004,279(36):37235-37240
Glutathione peroxidase (GPX) is one of the most crucial antioxidant enzymes in a variety of organisms. Here we described a new strategy for generating a novel GPX mimic by combination of a phage-displayed random 15-mer peptide library followed by computer-aided rational design and chemical mutation. The novel GPX mimic is a homodimer consisting of a 15-mer selenopeptide with an appropriate catalytic center, a specific binding site for substrates, and high catalytic efficiency. Its steady state kinetics was also studied, and the values of k(cat)/K(m)(GSH) and k(cat)/ K(mH(2)O(2)) were found to be similar to that of native GPX and the highest among the existing GPX mimics. Moreover, the novel GPX mimic was confirmed to have a strong antioxidant ability to inhibit lipid peroxidation by measuring the content of malondialdehyde, cell viability, and lactate dehydrogenase activity. Importantly, the novel GPX mimic can penetrate into the cell membrane because of its small molecular size. These characteristics endue the novel mimic with potential perspective for pharmaceutical applications. 相似文献