Rounds Of Biopanning Research Articles

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 RCA 120 . 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.

936. Phage-Selected Motifs Internalize Via Multiple Endocytic Pathways and Facilitate Gene Delivery

Top of pageAbstract The ability to direct internalization of macromolecules into cells or across endothelial barriers efficiently has tremendous therapeutic implications. Several internalization motifs (IM) that exist in nature have been defined and exploited, including the third -helix of the Drosophila Antennapedia homeodomain and the HIV TAT protein. To investigate the general properties required for protein-based macromolecule internalization we constructed and performed peptide phage display (PPD) with T7 bacteriophage random 7-mer peptide libraries. Functional biopanning with the T7 libraries was performed on microvascular endothelial cells (EC) to select peptide motifs that could internalize phage. The titer of internalized phage increased through sequential rounds of biopanning and resulted in selection of phage expressing IM with a finite shared sequence relationship between charged, polar, and hydrophobic amino acids. Selected IM were synthesized as rhodamine-labeled free peptides and their internalization properties were investigated in EC. Internalization of IM was inhibited at 4 degrees, suggesting an energy dependent mechanism of internalization. Confocal data showed that the IM was internalized via at least two different endocytic pathways: clathrin-coated pits and lipid rafts. Treatment with inhibitors of clathrin-mediated and lipid raft endocytosis inhibited uptake of IM, consistent with the result from confocal study. Further, in vitro and in vivo studies indicated that precomplexing of adenovirus with the IM facilitated the degree of viral transduction into endothelial cells and across endothelial barriers. These data suggest that relatively short peptide motifs are capable of directing macromolecules to diverse internalization pathways. It may be possible to exploit these characteristics for targeted delivery of therapeutic macromolecules to cells and tissues, and potentially to develop more efficient methods of gene transfer.

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k on value and/or a low k off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen ( k on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen ( k off′), the effective antigen concentration ( C), the time for the binding process ( t b) and the time for the washing process ( t w). An optimum set of operating parameters ( C, t b and t w) for isolating phages displaying an antibody with a high k on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k on value and one displayed an antibody with a 30-fold greater k on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein–protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 μM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein–protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X 3CX 6(W/F)(Y/F)CX 2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a K D value of 1.9 μM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.

Schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library.

In an attempt to isolate and identify the antigenic epitopes on ferritin of Schistosoma japonicum (SjFer) and to test their protective potentiality against Schistosoma japonicum (S.j), polyclonal antisera against SjFer was prepared to screen a 12-mer random peptide library. Three rounds of biopanning were performed and resulted in an enrichment. Six peptides selected randomly from the third elute were all found to be positive by evaluating the binding to anti-SjFer sera by ELISA and Western blotting. Three amino acid sequences were deduced from the six phage clones by sequencing. When they were used to vaccinate mice, the three peptides could induce significant reduction in adult worms (26.7%, 20.4%, and 25.9%) as well as in liver eggs per gram (LEPG) (40.0%, 38.2%, and 40.8%). This result showed that three mimotopes on SjFer were obtained and they could induce significant protective efficacy against S.j.

Broadly cross-reactive mimotope of hypervariable region 1 of hepatitis C virus derived from DNA shuffling and screened by phage display library.

The hypervariable region 1 (HVR1) is the target of neutralizing antibodies but with isolate specificity. The aim of this study was to obtain immunogenic mimotopes of HVR1, which can react broadly with different HVR1 antibodies and could be one of the candidate immunogens in an effective vaccine against HCV. Thirty-one HVR1 cDNA fragments were digested by DNase I into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 was then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. Eight individual phages were selected after four rounds of biopanning against anti-HVR1. ELISA was carried out on immobilized purified phages, respectively, to detect their reactivity with a panel of sera. DNA sequences of the inserts were analyzed and compared with the consensus sequences defined by Puntoriero et al. [(1998) EMBO J 17:3521-3533]. The reactivity of the eight selected clones to the 30 sera were from 53.3 to 80%. Among these, phage 13 (ETYVSGGSAARNAYGLTSLFTVGPAQK, aa 384-410) reacted most broadly. None of the selected sequences encoded for peptides corresponded to known HVR1 from original viral isolates. The two high reactive phages had the similar amino acid sequences with the consensus, which might play a particular role in determining the frequency of reactivity. In conclusion, this study has used effectively DNA shuffling combined with phage display technology to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Mimotope 13 and 23 appeared to be most reactive immunologically and could be candidate immunogens. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.

Identification of anti-TNFα peptides with consensus sequence

Phage displayed peptide library was used to select tumor necrosis factor α (TNFα) binding peptides. After three sequential rounds of biopanning, some linear TNFα-binding peptides were identified from a 12-mer peptide library. A consensus sequence ( L/ M) HEL( Y/ F)( L/ M) X( W/ Y/ F), where X might be variable residue, was deduced from sequences of these peptides. The phages bearing these peptides showed specific binding to immobilized TNFα, with over 80% of phages bound being competitively eluted by free TNFα. To confirm the binding activity and to explore further functional properties, three peptides with typical structure were selected and expressed as GST-fused protein. These recombinant peptides effectively competed for [ 125I]TNFα binding to TNFR1 in a dose-dependent manner, with IC 50 from 10 to 160 μM. Furthermore, the GST-fused derivatives showed inhibitory effects on TNFα-induced cytotoxicity. Taken together, these data demonstrate that the TNFα-binding peptides are effective antagonists of TNFα and the deduced motif might be useful in development of novel low molecular weight anti-TNFα drugs.

Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments

Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10 7 independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Use of a Phage Display Library to Identify Oligopeptides Binding to the Lumenal Surface of Polarized Endothelium by Ex Vivo Perfusion of Human Umbilical Veins

Human endothelial-specific targeting peptides were identified by biopanning within freshly-obtained human umbilical cords. Umbilical veins were cleaned in situ and M13 phage display libraries were passed through the cords. Tightly bound phage were recovered following isolation of endothelial cells by collagenase digestion and homogenisation, allowing production of enriched phage libraries for subsequent rounds of panning. After five rounds of biopanning, five promising sequences were selected and the binding of the corresponding phage clones was compared in perfused umbilical veins. Each of these peptides showed substantial binding, although the clone encoding the heptapeptide KPSGLTY showed the greatest, some 89-times greater than insertless phage. Binding of this phage clone was examined to cells in vitro, where it demonstrated at least five-times greater binding to isolated human umbilical vein endothelial cells than to 911, SKOV3, B16F10 and Cos7 cells. These initial peptides may prove useful targeting agents for endothelial-selective delivery, and this powerful approach should be readily applicable to biopanning in a broad range of human vessels ex vivo.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BackgroundThe development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised.MethodsP. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database.ResultsFollowing four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library.ConclusionThe construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag.

Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes. Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified. There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0+/-1.6) % to (39.0+/-2.7) %. Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Immunoscreening of phage-displayed cDNA-encoded polypeptides identifies B cell targets in autoimmune disease

Characterisation of self-antigens can contribute to an understanding of the aetiology of autoimmune disorders as well as to the development of new therapies and diagnostic methods. The present study was undertaken to investigate the applicability of complementary DNA (cDNA) phage-display technology to the identification of autoantigens recognised by the humoral response in autoimmune disease. Using systemic lupus erythematosus (SLE) as a model system, a pool of patient immunoglobulin G (IgG) was biopanned on a fibroblast cDNA phage-display library constructed in the vector pJuFo. Following three rounds of biopanning, recovered cDNAs were sequenced and then identified using BLAST comparisons with international databases. Both previously reported SLE autoantigens, for example, α-enolase and U1 small nuclear ribonucleoprotein-C (U1snRNP-C), and novel autoantibody targets, including ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), ubiquitin-like protein PIC1 (PIC1), and transcription factor-like protein MRG15 (MRG15), were recovered from the biopanning procedure. Radiobinding assays were used subsequently to confirm the reactivity of some putative autoantigens to panels of sera from SLE patients, control patient groups, and healthy individuals. SLE patient sera were positive for reactivity to: U1snRNP-C, 4/15 (27%); α-enolase, 1/15 (7%); RPS20, 3/15 (20%); RPS13, 1/15 (7%); PIC1, 1/15 (7%); and MRG15, 2/15 (13%). Overall, cDNA phage-display technology appears to be applicable to the identification of autoantigens in autoimmune disease.

Identification of FGF receptor-binding peptides for cancer gene therapy.

Linear FGF receptor-binding heptapeptides were identified by phage display using sequential rounds of biopanning against cells with displacement of phage by FGF2. The consensus motif MXXP was iterated after four to five rounds and the peptide MQLPLAT was studied in depth. Phage bearing MQLPLAT showed high levels of binding to FGF receptor positive cells, with over 90% of phage bound being eluted competitively by adding free FGF2. MQLPLAT phage showed only limited binding to Cos7 cells deficient in receptors for FGF. MQLPLAT phage bound to SKOV3 cells with a K(d) of 2.51 x 10(-10) M. Although binding could be blocked by preincubation with free FGF2, heparin could not displace the phage. Use of MQLPLAT to target polyelectrolyte gene delivery vectors in vitro in the presence of serum achieved up to 40-fold greater transgene transduction than nontargeted vectors. MQLPLAT phage were administered into gastric carcinomas via the tumor-feeding artery immediately following resection from patients. The phage showed up to 9-fold more accumulation in the tumor than in adjacent regions of normal tissue, whereas control phage showed less than 2-fold. These peptides should provide useful ligands for specific delivery of gene therapy vectors to clinically relevant targets.

Discovery of a Stable Dimeric Mutant of Cyanovirin-N (CV-N) from a T7 Phage-Displayed CV-N Mutant Library

Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure–activity requirements for CV-N's antiviral properties.

Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin.

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Phages with high affinity to the S protein obtained from rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of biopanning. 9 different peptides from the enriched library were selected by ELISA. Circular dichroism (CD) spectra of the GST-S fusion protein with binding phages and non-binding phages showed that structure of the S protein was changed after it bound to each of these 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of S protein. Thus, those peptides might be used to develop plant resistance and disrupt virus transmission. 3 of the 12-mer peptide genes were fused with the GST gene in pGEX 3X. The fusion proteins were also obtained usingE. coli expression system and purified.

Selection of peptide ligands binding to the basolateral cell surface of proximal convoluted tubules

Recently, we have reported a novel approach of screening phage-display peptide libraries on microdissected intact renal tubular segments and identified an RGD-containing peptide ligand that specifically binds to the basolateral membrane of cortical collecting ducts (CCD). However, screening phage libraries on proximal convoluted tubules (PCT) did not yield a tubule segment-specific ligand. Here, we describe the successful modification of our previously developed phage-display approach and the identification of two distinct ligands that bind specifically to receptors expressed at the basolateral membrane of PCT. Ex vivo screening of phage-display peptide libraries for specific ligands was adapted for PCT. The previously developed method was significantly extended by applying it to a distinct tubular segment, varying the number of rounds of biopanning and incubating phage libraries with absorber cells prior to biopanning. Binding specificity and cellular localization of selected peptide-displaying phage or the corresponding synthetic peptide were analyzed using various epithelial cell lines as well as competition assays and confocal immunofluorescence microscopy. Screening phage-display peptide libraries, depleted of ligands binding to ubiquitously expressed receptors by preincubation with HEK-293 cells, led to the identification of two PCT-specific ligands. Phage expressing peptides with the consensus sequence GV(K/R)GX3(T/S) or RDXR mediated 15-fold and 13-fold higher binding to PCT than control phage, and binding to PCT was 13-fold and 21-fold higher than binding to CCD, respectively. Neither phage mediated significant binding to various epithelial cell lines, and binding of both ligands was abolished by the addition of the corresponding synthetic peptide. Immunofluorescence experiments revealed a submembrane localization of both ligands upon incubation with PCT. Exploiting the versatility of phage-display and biopanning allowed the identification of two distinct peptide ligands that bind specifically to the basolateral membrane of PCT. Tubule segment-specific ligands, such as the described PCT ligands, may be useful for the analysis of cell-extracellular matrix interactions and may contribute to the development of new therapeutic strategies for renal diseases.

A mimotope of pre-S2 region of surface antigen of viral hepatitis B screened by phage display.

To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.