从随机肽库中筛选抗入ANG抗体的模拟小肽

目的从随机噬菌体十二肽库中寻找抗重组人 ANG抗体的模拟肽。方法以重组人 ANG为目标分子 ,对随机十二肽库进行亲合筛选 ,用噬菌体 EL ISA及竞争抑制实验鉴定阳性克隆的结合性和特异性。结果经过 3轮亲和筛选 ,噬菌体的回收率从 2 .0× 10 - 4 %增加到 9.3× 10 - 2 % ,阳性克隆得到富集 ;EL ISA和竞争抑制实验证明 :有 9个噬菌体克隆与靶分子有较强的结合活性 ,其中 6个阳性噬菌体克隆能特异性地抑制抗人重组 ANG单抗与 ANG的结合。结论该小肽可能是抗人ANG抗体的模拟小肽 ,可作为 ANG的拮抗剂     

用噬菌体表面显示肽库技术筛选乙酰胆碱酯酶有效抗原表位的研究

目的：筛选和鉴定乙酰胆碱酯酶的有效抗原表位。方法：收集乙酰胆碱受体抗体阴性而乙酰胆碱酯酶抗体阳性的重症肌无力病人血清，采用溴化氰活化的琼脂糖柱（CNBr-actived sepharose^TM4B）纯化抗乙酰胆碱酯酶的多克隆抗体并定量；用纯化的抗乙酰胆碱酯酶的抗体对随机的12肽噬菌体表面显示肽库进行5轮免疫学筛选，随机挑取克隆；采用Western blot免疫识别，识别为阳性的克隆进行核苷酸序列的测定，并与乙酰胆碱酯酶的氨基酸序列进行同源性比较。将获得的不同表位的阳性克隆分别免疫小鼠，采用Western blot筛选能刺激小鼠产生抗乙酰胆碱酯酶抗体的阳性克隆，进行生物学活性的鉴定。结果：经5轮免疫学筛选后挑取的49个克隆中，经Westem blot识别15个克隆能被抗乙酰胆碱酯酶抗体识别。核苷酸序列分析发现共有7种不同的表位，其中1种表位与乙酰胆碱酯酶有较高的同源性，其余6种表位与其无一级结构的同源性。经动物免疫初步实验筛选，共有5个免疫原性较强的阳性克隆，其免疫的鼠血清均可识别人乙酰胆碱酯酶。结论：获得了5种乙酰胆碱酯酶的有效抗原表位，1种可能为结构表位，4种为模拟表位。     

日本血吸虫病患者血清对噬菌体随机7肽库的免疫筛选

目的 从噬菌体随机多肽文库中,筛选出能与慢性血吸虫病患者血清特异性结合的短肽分子。方法 采用慢性血吸虫病患者血清球蛋白作为配基,免疫筛选以融合蛋白形式在丝状噬菌体Ｍ１３外壳蛋白Ⅲ表达的随机７肽文库。按吸附－洗脱－扩增的淘筛过程,经３轮免疫淘筛后,随机挑取菌体克隆用ＥＬＩＳＡ检测其牧场 划性,并用斑点ＥＬＩＳＡ分析其诊断血吸虫病的价值。结果 经３轮免疫淘筛后,特异性结合的噬菌体富集增加近１００倍,有     

用噬菌体肽库筛选重组日本血吸虫线粒体相关蛋白的表位

目的：筛选和鉴定重组的日本血吸虫（中国大陆株）线粒体相关蛋白rSj38的表位。方法：用纯化的rSj338/26GST免疫家兔获得抗rSj338/26GST的多克隆抗体IgG，将抗体进一步纯化，获得抗rSj338单特异多克隆抗体IgG。用纯化抗rSj338抗体对噬菌体12肽库进行5轮免疫学筛选，挑取克隆。采用Western blot免疫识别，核苷酸序列测定分析其获得的表位并与rSj338/26GST进行同源性比较。将获得的不同表位的阳性克隆分别免疫小鼠，并采用Western blot及dot-ELISA方法筛选能刺激小鼠产生较高滴度抗rSj338抗体的阳性克隆，并将阳性噬菌体免疫的小鼠血清对纯化的rSj338/26GST,26GST，日本血吸虫成虫及虫卵抗原进行Western blot识别。结果：经5轮免疫学筛选后挑取的32个克隆，用Western blot方法30个克隆能被抗rSj338抗体识别，核苷酸序列分析发现共有11种表位，与rSj338无一级结构的同源性。经动物免疫初步实验筛选。共获得4个免疫原性较强的阳性克隆，其免疫鼠血清均可识别rSj338/26GST，日本血吸虫成虫及虫卵抗原。结论：获得了4种日本血吸虫中国大陆株线粒体相关蛋白rSj338的表位，均为模拟表位，这将为日本血吸虫病的疫苗研究开辟新的途径。     

Functional inhibition of CCR3-dependent responses by peptides derived from phage libraries.

So far chemokine antagonists have been identified by modification of the NH2-terminus of known chemokines or by screening large number of compounds in functional assays. Here we used phage display peptide libraries to identify hexapeptides that antagonize the interaction between eotaxin and its receptor CCR3. The peptide sequence CPWYFWPC was recovered by panning phage libraries on CCR3-transfected murine pre-B cells after elution with eotaxin. The synthetic, structurally constrained peptide effectively competed 125I-eotaxin binding to CCR3 (IC(50) = 20 microM). Furthermore, it had weak agonistic effects on Ca(2+) mobilization in CCR3 transfectants that underwent heterologous desensitization by subsequent exposure to eotaxin. The peptide inhibited chemotaxis of CCR3 transfectants induced by a broad panel of CCR3 ligands. Specificity was tested with the CXCR1, CXCR2, CXCR3 and CCR5 receptors. In experiments aimed at characterization of residues necessary for eotaxin binding, we affinity purified the linear eotaxin-binding peptide VTPRQR, and showed that the peptide displaced the binding of radiolabeled eotaxin to CCR3 (IC(50) = 300 microM) ina dose-dependent manner, inhibited eotaxin induced increases in intracellular Ca(2+), and migration of CCR3-transfected cells. Specificity was affirmed using other CCR3 ligands. This is the first de novo identification of chemokine antagonists by direct screening on target proteins.     

用噬菌体随机肽库筛选刺激细胞生长的活性小肽

目的 筛选刺激NFS－60细胞生长的活性小肽。方法 以G－CSF依赖细胞系NFS－60为靶细胞，筛选随机6和15肽库，经3轮筛选后，随机挑取60个噬菌体克隆进行生物学活性和结合活性鉴定。结果 得到6个刺激NFS－60细胞生长的阳性噬菌体克隆。经细胞ELISA检测，这6个小肽都可与NFS－60细胞结合，并且这6个小肽与细胞结合的量，都与所加入的噬菌体的量成正比，经测序未找到共同序列。结论 从随机噬菌体肽库中筛到的活性小肽，可以模拟某些生长因子的活性部位，来刺激靶细胞的生长。     

Rapid and efficient identification of epitopes/mimotopes from random peptide libraries

Phage-displayed random peptide libraries are important tools in identifying novel epitopes/mimotopes that may lead to the determination of antigen specificity. In this approach, high-affinity phage peptides are enriched by affinity selection (panning) on a monoclonal antibody. To facilitate identification of all potential phage peptides specific for recombinant monoclonal antibodies (rAbs) previously generated from clonally expanded plasma cells from the cerebrospinal fluid of patients with multiple sclerosis (MS), we developed a high-throughput method to determine phage specificity. In contrast to the 8-9 days needed in the standard large-scale method of amplifying phage clones for ELISA, the high-throughput method takes only 1 day. ELISA using phage clones amplified directly in 96-well plates avoids large-scale phage purification and enables rapid identification of specific epitopes/mimotopes. This technique will expedite identification of MS-specific peptides that can be used to discover the corresponding protein antigens.     

Neutralizing peptide ligands selected from phage-displayed libraries mimic the conformational epitope on domain III of the Japanese encephalitis virus envelope protein

The envelope (E) protein of Japanese encephalitis virus (JEV) contains 500 amino acids with six "conserved" disulfide bonds to maintain its conformational structure. Neutralizing epitopes located on the E protein are mostly conformational dependent. In this study, we used phage-displayed 12-residue combinatorial peptide libraries to select high-affinity peptide ligands bound to monoclonal antibody E3.3. The specific peptide ligands presented on ten high-affinity phage clones displayed six different amino acid sequences, all showing a novel cis-proline turn structure. After being superimposed onto the best fit of the three-dimensional structure of JEV E protein, these peptide structures were mapped to a conformational region constituted by three continuous polypeptide segments (E307-E309, E327-E333, E386-E390) in domain III. Synthetic peptide ligands based on one peptide sequence (E18) were further investigated using alanine scanning within the cis-proline turn structure to demonstrate its unique molecular characteristics. Our results showed that three residues forming the novel cis-proline turn structure were all important in eliciting JEV-specific neutralizing antibodies in mice.     

Characterization and identification of allergen epitopes: recombinant peptide libraries and synthetic, overlapping peptides.

For the understanding of the relationship between protein structure and allergenicity, it is important to identify allergenic epitopes. Two methods to characterize primarily linear epitopes are compared using the major allergen from brown shrimp (Penaeus aztecus), Pen a 1, as an example. A recombinant peptide library was constructed and synthetic, overlapping peptides, spanning the entire Pen a 1 molecule, were synthesized and tested for specific IgE reactivity. Both methods identified IgE-binding of Pen a 1, however, the SPOTs procedure resulted in the identification of more epitopes of the major shrimp allergen Pen a 1 than the usage of the recombinant peptide library. For detection of specific IgE antibodies, the usage of 125I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies. The regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. If a given food allergen contains significant linear epitopes, which seems to be true for stable major allergens such as those of peanut and shrimp the SPOTs system may be more advantageous than the use of recombinant peptides libraries. However, if allergens are studied that contain more conformational epitopes, recombinant peptide libraries may help to identify the relevant epitopes. It has to be emphasized that no system for epitope identification will detect all epitopes and that the relevance of identified epitopes has to be confirmed with other methods such as inhibition studies, crystallographic analysis or the immunological evaluation of modified whole allergens.     

An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries

Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies. It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immuno-precipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution. The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive.     

Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases.

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4 h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15 kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137C1 by these sera. In addition, MoAb 137C1 and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137C1-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.     

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV-1 envelope protein gp120LAI, and has previously been shown to interact with the -I-GPGRA- motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif -GPGRA-, a similar segment, -GPAR-, and two unrelated motifs -FRLLG- and -WRM/ALG- were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide-specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the -GPGRA- containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV-1SF2 virus in vitro.     

Selection of phage displayed peptides from a random 10-mer library recognising a peptide target

Background: Peptide display libraries are powerful tools in the search for detailed information about protein–protein interactions. Usual targets for isolation of phage displayed peptide ligands include antibodies, various receptors, other full size proteins or larger fragments thereof. Smaller protein fragments such as synthetic peptides have not been reported as targets for screening of peptide display libraries. Objectives: To investigate whether a protein target used for screening of a peptide display library could be scaled down to peptide size. As the peptide target we wanted to use a sequence derived from the cytosolic tail of MHC class II associated invariant chain containing a leucine class endosomal sorting signal, known to be recognised as an autonomous functional unit during targeting of class II complexes to antigen processing compartments. Study design: A screening procedure where a synthetic 15-mer invariant chain peptide was coupled to a methacrylate matrix of high binding capacity was developed, and three rounds of selection were performed from a random 10-mer fUSE5 display library. Results: The peptide display library was successfully enriched for phage clones with affinity for the invariant chain peptide. Furthermore, the binding phage clones were able to distinguish between a functional and a mutated form of the target. These clones therefore displayed possible peptide mimetics of signal recognition sites in the cellular sorting machinery. Conclusion: The size of a protein target may be scaled down to peptide size and be recognised by a 10-mer peptide displayed on filamentous phage. This approach may particularly be useful when the peptide target contains a functional unit for recognition.     

Multiple antigen peptides for specific detection of antibodies to a malaria antigen in human sera.

Multiple antigen peptides (MAP), consisting of a number of peptide copies synthesized on a branching lysyl core, offer a novel approach for rendering small peptides immunoreactive in solid-phase immunoassays. An octameric MAP, carrying 6 repeats of the sequence -N-A-A-G-, tandem repeated in the immunodominant region of the circumsporozoite (CS) protein of Plasmodium malariae, was used as a model to evaluate the suitability of the MAP system in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against a parasite antigen in individuals exposed to natural infection. The reaction of endemic sera in ELISA on MAP8-(NAAG)6 was related to that obtained in immunofluorescence on sporozoites, indicating the specificity of the antibody-MAP interaction. The reactivity of immune sera was found to be directed only against the (NAAG)6 moiety of the MAP and not against the lysyl core, since antibody binding to MAP8-(NAAG)6 was completely inhibited by (NAAG)6-NA monomer, but remained uninfluenced when lysyl core was used as competing ligand. The levels of antibodies to MAP8-(NAAG)6, in two groups of individuals naturally exposed to malaria infection, appeared to be related to their respective exposure to the parasite.     

Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera.

Nine oligopeptides corresponding to segments of different open reading frame (ORF) proteins of human papillomavirus (HPV) 6b and HPV-16 were prepared and tested for reactivity with human sera in enzyme-immunoassay (ELISA). Of these only heptadecapeptide derived from L2 ORF of HPV-6b, and encoded also by L2 ORF of HPV 11, was reactive with some human sera. Over 400 human sera of different origin were tested for the presence of antibody to this antigen. While less than 15% of sera from healthy subjects or cervical carcinoma patients were found antibody positive, sera from the majority of condylomata accuminata (CA) patients were reactive. The antibody titres varied from 1:10 (initial serum dilution) to 1:80; in this respect there was no marked difference between sera from CA patients and the other subjects. The prevalence of antibody was higher among promiscuous than nonpromiscuous women. This is in line with the assumption that sexual intercourse is the most important route of HPV 6 and 11 transmission.     

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.     

KCTD9多肽抗体的制备和应用

目的:利用人工合成多肽制备针对人源、鼠源KCTD9(hKCTD9、mKCID9)的特异性多克隆抗体,以期用于与KCTD9表达异常密切相关疾病的研究和诊治.方法:根据hKCTD9、mKCTD9基因编码的氨基酸序列合成多肽(Pep),并用化学方法与匙孔血蓝蛋白(KLH)连接,纯品Pep-KLH免疫纯种新西兰雄性大白兔,所制备的抗血清用ELISA、Western blot实验鉴定,并采用免疫组化法应用于正常人和乙型肝炎患者肝组织中hKCTD9的检测.结果:ELISA检测表明,用Pep-KLH免疫的大白兔所制备的抗血清可与Pep发生特异性免疫反应;Western blot结果显示,抗血清可识别MHV-3感染BALB/cJ小鼠PBMC特异性条带,相对分子质量(Mr)为37 KD.对正常人和病毒性乙型肝炎患者肝组织免疫组织化学染色发现hKCTD9在正常人和重型乙型肝炎患者均有表达,但是在重型乙型肝炎患者高表达.结论:所制备的KCTD9的多肽抗体(多克隆抗体)可识别mKCTD9和hKCTD9分子,具有与抗原特异性结合,为深入研究这一新发现的分子提供了有力的科学手段.     

Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin

We designed hybrid peptides, SIRVXVXPG (X: A or G), from a laminin-derived peptide, SIKVAV, and an elastin-derived peptide, VGVAPG, and tried to develop new alginate dressings linked covalently with the hybrid peptides. First, we examined the effectiveness of the hybrid peptides for cell attachment and proliferation using normal human dermal fibroblasts (NHDF) in vitro. The hybrid peptides promoted attachment of NHDF, whereas neither Ac-KSIKVAV nor Ac-KVGVAPG promoted attachment. Although all the peptides we examined promoted the proliferation of NHDF to some extent, the hybrid peptide-coated plates showed strong NHDF proliferative activity, compared with the other peptide. Next, we created alginate dressings linked with some of these peptides and examined their effectiveness in wound healing using a rabbit ear skin defect model in vivo. Nine days after operation, ears with the alginate dressings linked with the hybrid peptides showed significantly greater epithelialization and a larger volume of regenerated tissue compared to those treated with SIVAV-linked, VGVAPG-linked and unlinked alginate dressings. These new alginate dressings linked with the hybrid peptides could be promising dressings especially for wounds with impaired healing.     

Structural aspects of small peptides. Synthesis and characterization of protected homo-oligomers derived from L-alanine

A series of protected L -alanine homo-oligomers from dimer to heptamer was prepared by the dicyclohexylcarbodiimide and acyl azide coupling methods. In the course of the synthetic approach tert-butoxycarbonyl as the N-protecting and methyl ester as the C-protecting end groups were employed. The monodisperse oligomers prepared in this manner were characterized and found to be chemically pure. A polarimetric investigation in a structure disrupting solvent also indicated that all reactions proceeded with complete retention of the configuration. In addition, the dependence of the experimental molar rotation values at 589 nm of L -alanine internal residues in a polypeptide in a statistical coil conformation on the end groups of the molecule and the solvent is emphasized.     

Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera.

The ability of synthetic peptides encompassing almost the entire sequence of snRNP U1A polypeptide to be recognized in ELISA by sera of autoimmune patients was investigated. Sera from 18 patients with mixed connective tissue disease (MCTD), 145 with systemic lupus erythematosus (SLE) and 120 with other rheumatic autoimmune diseases were tested with 13 overlapping peptides. Among them, peptide 257-282 and, to a lower extent, peptide 1-11 were recognized by MCTD, SLE and Sjögren''s syndrome sera. In contrast, peptide 35-58 was recognized by 94% of MCTD and only 19% of SLE sera. It did not react with any of the other patient sera. The ELISA results were compared with the pattern of reactivity observed in immunoblotting. The results indicate that peptide 35-58 probably contains a major epitope recognized by MCTD autoantibodies. It is noteworthy that in snRNP particles, this region of U1A interacts with RNA and presents only limited homology with the corresponding sequence 32-50 of U2B''''.