A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb₃-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb₃ antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb₃ antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb₃ antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb₃ mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb₃Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb₃ mimic peptide potentially has great promise for use against Stxs.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

Self-assembled monolayers of globotriaosylceramide (Gb3) mimics: surface-specific affinity with shiga toxins

Self-assembled monolayers (SAMs) of Gb3 mimics having different lengths of alkyl chains were prepared on gold surfaces, and their interactions with galactose-specific lectin (RCA(120)) and Shiga toxins (Stxs) were investigated by a quartz crystal microbalance (QCM) in aqueous solutions. Their interaction with RCA(120) was enhanced owing to the "cluster effect," regardless of the alkyl chain length of the SAMs. The interaction with Stxs was dependent on the alkyl chain length of Gb3 mimics. Stx-1 and Stx-2 showed a stronger affinity to the Gb3C2 SAM with ethyl disulfide and to the Gb3C10 with decyl disulfide, respectively. Gb3 glycoconjugate polymer with no alkyl spacer inhibited the adsorption of Stx-1 to Gb3C10 SAM but did not inhibit the adsorption of Stx-2 to Gb3C10 SAM. The results suggest that the alkyl chain of the glycolipid takes part in the binding to Stx-2 but not to Stx-1, which is also supported by the computer simulation of Stx-1 with a Gb3 model substance.     

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.     

噬菌体展示技术筛选bFGF模拟短肽

目的：通过噬菌体展示技术筛选得到与FGFR结合的bFGF模拟短肽,为bFGF肽类抑制剂的研发提供实验基础。方法：以Balb/c 3T3细胞为靶标,以COS-7细胞作消减,对噬菌体随机七肽库进行4轮生物淘洗,再采用ELISA检测单克隆噬菌体对Balb/c 3T3亲和性和特异性,选取阳性克隆进行DNA测序分析。结果：从富集的噬菌体中获得12个阳性克隆,获得一组疏水性七肽及共同基序PR。结论：利用肽类新药开发的重要工具--噬菌体展示技术,得到2段bFGF的受体结合模拟肽,可望作为bFGF抑制剂的先导肽。     

Inhibitory effect of intestinal anti-Gb3 IgA antibody on verotoxin-induced cytotoxicity

The effect of intestinal IgA antibody against the receptor for verotoxin (VT), globotriaosylceramide (Gb3), on VT-mediated cytotoxicity was examined. Intestinal IgA antibodies against Gb3 were prepared by oral immunization of mice with Gb3 and adjuvant monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). Oral administration with Gb3 and MPL-containing PS-liposome induced significant IgA responses to Gb3 in the intestinal lavage fluid in all mice tested. Furthermore, anti-Gb3 IgA antibodies in the lavage fluid effectively inhibited the cytotoxicity of VT2 to Vero cells in a dose-dependent manner. These results suggest that anti-Gb3 IgA antibodies produced in the intestinal tract, upon oral immunization with Gb3-containing liposome, function as inhibitors against VT and also indicate the potential usefulness of oral PS-liposome vaccines containing MPL for the induction of a protective mucosal immune response against intestinal diseases.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.     

Characterization of a shiga-toxin 1-resistant stock of vero cells

Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1-resistant (R-) stock of Vero cells. (1) When the susceptibility of R-Vero cells to Stx1 cytotoxicity was compared to that of Stx1-sensitive (S-) Vero cells by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, cell viability after 48-hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence-labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti-Gb3Cer monoclonal antibodies capable of binding to S-Vero cells failed to effectively label R-Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R-Vero cells. (3) The lipid analysis also showed that the R-Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S-Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R-Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R-Vero and S-Vero cells. Further study of R-Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.     

利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)颗粒筛选噬菌体肽库,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。【方法】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

利用噬菌体随机肽库筛选抗柯萨奇病毒B3多肽的研究

本实验将柯萨奇病毒B3型(CVB3)大量扩增,应用蔗糖密度梯度离心法纯化病毒。利用噬菌体随机9肽库进行筛选,3轮淘洗后,测定噬菌体克隆抗病毒复制能力。提取阳性克隆DNA并进行测序,推导外源多肽的氨基酸序列。结果表明:3个具有明显抗病毒复制能力的噬菌体阳性克隆被筛选出来,使TCID50由10-7.5SFU/mL分别降至10-5.25、10-6、10-5.5SFU/mL,由此证明可以应用噬菌体肽库来筛选具有抗病毒作用的多肽,本研究为抗病毒多肽制剂的研究奠定了基础。     

利用噬菌体展示技术筛选胰岛素模拟肽

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.     

犬种布鲁氏菌ZG株单克隆抗体4H3抗原模拟表位的筛选及分析

【背景】目前犬布鲁氏菌病诊断存在一定的困难。【目的】筛选并研究犬种布鲁氏菌单克隆抗体4H3株的特异性抗原表位。【方法】利用噬菌体肽库展示技术,以犬种布鲁氏菌单克隆抗体4H3株作为靶分子,包被酶标板,用12肽随机肽库经过3轮生物淘洗程序进行筛选。经过3轮筛选后,噬菌体产出率从5.00×10^-7增加到9.84×10^-6,假阳性率逐轮降低。从第3轮筛选的阳性克隆中随机挑取14个进行增殖,提取基因组DNA,进行测序分析;并通过iELISA和cELISA检测阳性克隆的亲和性和特异性。【结果】14株阳性单克隆噬菌体共出现3种不同的短肽序列,分别是KMSIRHPIRLPI、ILRRRRKRIIQI和QRIHMRLTTQS;iELISA结果表明3种短肽序列与单克隆抗体的亲和性依次为KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS;cELISA结果显示短肽KMSIRHPIRLPI和ILRRRRKRIIQI特异性较强。对亲和性较强、特异性较高的2条短肽KMSIRHPIRLPI和ILRRRRKRIIQI展开具体分析,比对分析表...     

Humoral immune response against proteophosphoglycan surface antigens of Entamoeba histolytica elicited by immunization with synthetic mimotope peptides

The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.