Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.

Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide （WH-16） was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.

PSMA mimotope isolated from phage displayed peptidelibrary can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cellsurface glycoprotein expressed predominantly in prostatesecretory acinar epithelium and prostate cancer cells aswell as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domainof PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5reactive phagotopes were identified. Sequence analysis ofisolated clones demonstrated that the interaction motif'VDPA/SK' has high homology to 719-725aa on PSMA.Immunohistochemical staming of the prostate cancer sam ple with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotopeisolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.

Screening and Identification of a Targeting Peptide to nGLP-1R from Phage Display Peptide Library

In order to provide the structure information for designing new exendin-4 analogues, a phage display peptide library was screened by targeting the N-terminal extracellular domain of GLP-1R（nGLP-1R）. After four rounds of selection, nine sequences were obtained, four of them have higher affinity for nGLP-1R than the others. We chose two of them named X and Y peptides. Islet β-cell proliferation assay suggested that X and Y peptides didn＇t have any activity to increase islet β-cell proliferation. In other words, X and Y peptides were not agonists to GLP-1R. However, the conservative motifs of X and Y peptides provided us useful information to design new exendin-4 analogues.

Screening and identification of mimotopes of LPS conservative epitope from random phage display peptide library

Objective: To screen and identify the mimotopes of lipopolysaccharide (LPS) epitope. Methods: A random phage display dodecapeptide library was screened with monoclonal antibody 2B4 specifically against LPS conservative epitope. The positive clones were identified by phage EUSA and competitive inhibition assay with either S. typhimurium T861 LPS or E. coli Olll:B4 LPS. Results: After 3 rounds of biopanning, the clones bound with monoclonal antibody 2B4 were well enriched with the positive rate of 80%. The bindings between 12 positive phage clones and screening antibody were inhibited by both kinds of LPS. The positive peptide sequences were deduced from the corresponding DNA sequences. There were some identical sequences among them: PPQWFFSQPQL (5/12, 41. 7%), LPQYFW NTATTA (3/12, 25%), FPQNHWNVPWAT(2/12, 16. 6% ),HSQSFWNAPLAM and AHPWTHGYFPPL (l/12, 8. 3% ). Conclusion: The peptides screened with 2B4 antibody are mimotopes of LPS conservative epitope.

Screening of TACE Peptide Inhibitors from Phage Display PeptideLibrary

Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （TS00） and full-length ectodomain （T1300） of TACE were amplified by RT-PCR, and the expres.sion plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant TS00 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that TS00 and T1300 were highly expressed in the form of inclusion body. After Ni^2＋-NTA resin affinity chromatography, the recombinant proreins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence （TRWLVYFSRPYLVAT） was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells （PBMC） up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE.

Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.

Construction of Human ScFv Phage Display Library against Ovarian Tumor

In order to construct a single chain fragment variable （ScFv） phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes （VH） and light chain variable region genes （VL） were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 10^9 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 μ 10^12 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Construction of white spot syndrome virus (WSSV) whole genome phage display library

A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus （WSSV） genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ～2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 10^5.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ～ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

Selection of the specific coalescent peptide of human CD59 by phage display techniques

To screen and identify the short peptides with specific binding activity to human CD59 and to design the short-peptide clamp against tumor escape, the phage display peptide library containing 12 peptides was used to select the highly expressed specific coalescent peptide of human CD59 in CHO cells. Positive phage clones obtained after 5 rounds of biopanning and detected with ELISA were obtained, in which 8 of them with high binding activity to human CD59 were sequenced. The 3 sequences thus obtained showed high homology with each and certain homology with sequence with human CD2 （PubMed 339HGAAENSLSPSS）, and all contained primary structure , of which this sequence may be the mimic confonnational epitope binding to human CD59. These results in the present study may be helpful to design the short-peptide clamp against the active sites of CD59 on tumor escape.

Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

Staphylococcus aureusβ-hemolysin-neutralizing single-domain antibody isolated from phage display library of Indian desert camel

Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.

Affinity peptide developed by phage display selection for targeting gastric cancer

AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal-appearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV(AAD) appeared in 25%(12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically signif icant(P < 0.01) for gastric cancer as compared with other histological classif ications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus(ASFV)is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies(mAbs)and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein(p72)single-chain antibody and fused with an IgG Fc fragment(scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide(221MTGYKH226)of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.

MAPPING EPITOPES OF HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN WITH A PHAGE DISPLAY EPITOPE LIBRARY

The objective of this study is to map epitopes on HPMAPV16 L1 protein and provide information to the design of HPV16 prophylactic peptide vaccine. The epitopes on L1 protein were screenedby polyclonal and two monoclonal antibodies (BS and F4G3) against RPV16 L1 protin from a 6-merfd phage display epitope library with the method or immuuo-afrinity screening (Biopauuing). Aferthree rounds or Bio-Panning, the Positive phages were detected by L1 antibodies again with ELISA.The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Three mimotopes have been screened by polycloual and two monoclonal antibodies. The mimotope(LSLFSC) reacted with mouoclonal antibody B8 showed 50% pomology with the sequence 270275a. a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) reacted with polyclonalantibodies had 66% pomology with the L1 sequence 516~521a. A(TTSSTS), also a mimotope (DRWDRF) was found had the bomologic RF with the known L1 sequence 441 ~446a. a. The mimotopesLSLFSC and DRWDRF were adjacent to the epitopes at 267~269a. a and 422~441 a. a reported byother researchers Previously. Our results suggest that there might be a batch of epitopes on HPV16L1 ppotein, and the predominant epitopes of HPV16 L1 protein are located in the above two domains. These results will be helpful for design or HPV16 prophylactic peatide vaccines and HPVpolyvalent vaccines.

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and isolated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.

Selection of the Mimic Epitopes of Antigens from Schistosoma japonicum by Using Phage Display Techniques

To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide library of 12 amino acids were screened with purified IgG from normal rat sera. Positive clones that were obtained after three rounds of biopanning were detected by ELISA, and two of them were sequenced. Kunming mice were immunized with mixed phage clones. Each mouse was challenged with 40±1 S.j cercariae, and all mice were perfused 45 days post-challenge. The worms and the liver eggs were counted. The results were that the specific phages binding to IgG were enriched 300 folds after three rounds of biopanning. Twenty clones were detected by ELISA and 19 of them bound to the specific IgG of rat sera. The sequence of two clones revealed no homology with other sequences in the GenBank. Compared with the control groups, the reduction rates of the worm burden and liver egg were 33.6% and 59.8%, respectively. It was concluded that the specific peptides, which simulate antigenic molecules correlated with natural resistance to S.j in rats could be obtained by immunosceening phage random peptide library and a protective immunity against S.j can be detected by these epitopes in mice.

Application of phage display technology in targeted therapy of breast cancer

Phage display is a technology of gene expression and screening, it is widely used in the fields of defining antigen epitopes, signal transduction, genetic treatment, parasites research and tumor targeted therapy. Breast cancer is the most common cancer in women, we can obtain peptides specially associated with breast cancer by using phage display technology, and this method has great potential in early diagnosis of breast cancer and development new targeted drugs.