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 cellula...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.展开更多
Peptide-based therapeutics are increasingly pushing to the forefront of biomedicine with their promise of high specificity and low toxicity.Although noncanonical residues can always be used,employing only the natural ...Peptide-based therapeutics are increasingly pushing to the forefront of biomedicine with their promise of high specificity and low toxicity.Although noncanonical residues can always be used,employing only the natural 20 residues restricts the chemical space to a finite dimension allowing for comprehensive in silico screening.Towards this goal,the dataset comprising all possible di-,tri-,and tetra-peptide combinations of the canonical residues has been previously reported.However,with increasing computational power,the comprehensive set of pentapeptides is now also feasible for screening as the comprehensive set of cyclic peptides comprising four or five residues.Here,we provide both the complete and prefiltered libraries of all di-,tri-,tetra-,and penta-peptide sequences from 20 canonical amino acids and their homodetic(N-to-C-terminal)cyclic homologues.The FASTA,simplified molecular-input line-entry system(SMILES),and structure-data file(SDF)-three dimension(3D)libraries can be readily used for screening against protein targets.We also provide a simple method and tool for conducting identity-based filtering.Access to this dataset will accelerate small peptide screening workflows and encourage their use in drug discovery campaigns.As a case study,the developed library was screened against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)main protease to identify potential small peptide inhibitors.展开更多
Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rou...Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.展开更多
AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis...AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6).The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.展开更多
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 monoc...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.展开更多
When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to st...When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.展开更多
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 f...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.展开更多
AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) ...AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.展开更多
Combinatorial peptide libraries have become powerful tools to screen functional ligands by the principle of affinity selection. We screened in a phage peptide library to investigate potential peptide affinity ligands ...Combinatorial peptide libraries have become powerful tools to screen functional ligands by the principle of affinity selection. We screened in a phage peptide library to investigate potential peptide affinity ligands for the purification of human granulocyte colonystimulation factor(hGCSF). Peptide ligands will be promising to replace monoclonal antibodies as they have advantages of high stability, efficiency, selectivity and low price.展开更多
A library of 2 ×107 random octapeptides was constructed by use of phagemid-based monovalent phage display system. The randomly synthesized degenerated oligodeoxyribonucleotides (oligos ) were fused to the truncat...A library of 2 ×107 random octapeptides was constructed by use of phagemid-based monovalent phage display system. The randomly synthesized degenerated oligodeoxyribonucleotides (oligos ) were fused to the truncated g Ⅲ (p230-p403). Sequence analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (aa) sequences are randomly distributed. The library was used to select binding peptides to the monoclonal antibody (mAb) 9E10, which recognizes a continuous decapeptide epi- tope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequence analysis of the selected positive clones indicated that the binding sequences could fall into two classes, one class (clone 1) shares a consensus motif, ISE x x L, with c-myc decapeptide; and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically inhibited by free c-myc decapeptide. The immunogenicity of the phage peptide was further investigated by construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either clone could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected peptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.展开更多
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 round...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.展开更多
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-m...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.展开更多
AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolate...AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.展开更多
Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study ...Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.展开更多
Objective To obtain peptide mimicking epitopes of Schistosoma japonicum (S.japonicum) through screening of a phage peptide library and to test their potential for induction of protection. Methods S.japonicum infect...Objective To obtain peptide mimicking epitopes of Schistosoma japonicum (S.japonicum) through screening of a phage peptide library and to test their potential for induction of protection. Methods S.japonicum infected sera from Microtus fortis (IMFS) and normal sera from Microtus fortis (NMFS) were used respectively to screen a 12-mers random peptide library by testing the reactivity of anti-S.japonicum serum with the phagotopes. After three rounds of biopanning, the pooled phages were used to immunize mice, after which challenge infection was performed. Results Of 12 randomly picked clones, 10 clones selected using IMFS and 7 clones selected using NMFS were shown to be antigenic. Significant reduction in adult worms (22.6%) and a high reduction (68.9%) in liver eggs were achieved following immunization with phages screened with IMFS. However, no protection was elicited by those selected with NMFS. Conclusion The results show that the phagotopes are both antigenic and immunogenic, suggesting a potential use of phage displayed peptide as novel vaccines against S. japonicum.展开更多
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 e...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.展开更多
In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-bindi...In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.展开更多
基金Supported by the National Natural Science Foundation of China (Grant No. 39900116)
文摘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.
文摘Peptide-based therapeutics are increasingly pushing to the forefront of biomedicine with their promise of high specificity and low toxicity.Although noncanonical residues can always be used,employing only the natural 20 residues restricts the chemical space to a finite dimension allowing for comprehensive in silico screening.Towards this goal,the dataset comprising all possible di-,tri-,and tetra-peptide combinations of the canonical residues has been previously reported.However,with increasing computational power,the comprehensive set of pentapeptides is now also feasible for screening as the comprehensive set of cyclic peptides comprising four or five residues.Here,we provide both the complete and prefiltered libraries of all di-,tri-,tetra-,and penta-peptide sequences from 20 canonical amino acids and their homodetic(N-to-C-terminal)cyclic homologues.The FASTA,simplified molecular-input line-entry system(SMILES),and structure-data file(SDF)-three dimension(3D)libraries can be readily used for screening against protein targets.We also provide a simple method and tool for conducting identity-based filtering.Access to this dataset will accelerate small peptide screening workflows and encourage their use in drug discovery campaigns.As a case study,the developed library was screened against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)main protease to identify potential small peptide inhibitors.
文摘Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.
基金Supported by the Natural Science Foundation of Guangdong Province,China, No. 980120the Foundation of Excellent Youth Teacher,China,2001
文摘AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6).The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.
文摘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.
基金Supported by the Technology Development Funds of Education Department of Jilin Province,China(No.2008110)
文摘When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.
基金a grant from National Natural Science Foundation of China(N0.30171062)
文摘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.
基金Supported by the WHO/TDR, No. 980255 and the Science Commission of Hunan Province, No. 00jzy2115
文摘AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.
基金Supported by the National Natural Science Foundation of China(No.29774010)
文摘Combinatorial peptide libraries have become powerful tools to screen functional ligands by the principle of affinity selection. We screened in a phage peptide library to investigate potential peptide affinity ligands for the purification of human granulocyte colonystimulation factor(hGCSF). Peptide ligands will be promising to replace monoclonal antibodies as they have advantages of high stability, efficiency, selectivity and low price.
文摘A library of 2 ×107 random octapeptides was constructed by use of phagemid-based monovalent phage display system. The randomly synthesized degenerated oligodeoxyribonucleotides (oligos ) were fused to the truncated g Ⅲ (p230-p403). Sequence analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (aa) sequences are randomly distributed. The library was used to select binding peptides to the monoclonal antibody (mAb) 9E10, which recognizes a continuous decapeptide epi- tope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequence analysis of the selected positive clones indicated that the binding sequences could fall into two classes, one class (clone 1) shares a consensus motif, ISE x x L, with c-myc decapeptide; and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically inhibited by free c-myc decapeptide. The immunogenicity of the phage peptide was further investigated by construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either clone could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected peptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.
文摘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.
文摘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.
文摘AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.
基金This study was supported by grants from the Nationa1 Natura1 Science Foundation of China （No. 30670571, 30772258 and 81071560）, Science and Technology Research Program of Shandong Province （No. 2009GG10002078）, Scientific Research Development Plan of the Department of Education of Shandong Province （No. J07WD03） and National Basic Research Program of China （973 Program） （No. 2005CB522603）.
文摘Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.
基金ThisworkissupportedbygrantsfromMinistryofScienceandTechnologyofChina (No 96 A2 3 0 6 0 4),WHO/TDR (IDNo 980 2 5 5 )andNewEnglandBiolabs,Inc USA
文摘Objective To obtain peptide mimicking epitopes of Schistosoma japonicum (S.japonicum) through screening of a phage peptide library and to test their potential for induction of protection. Methods S.japonicum infected sera from Microtus fortis (IMFS) and normal sera from Microtus fortis (NMFS) were used respectively to screen a 12-mers random peptide library by testing the reactivity of anti-S.japonicum serum with the phagotopes. After three rounds of biopanning, the pooled phages were used to immunize mice, after which challenge infection was performed. Results Of 12 randomly picked clones, 10 clones selected using IMFS and 7 clones selected using NMFS were shown to be antigenic. Significant reduction in adult worms (22.6%) and a high reduction (68.9%) in liver eggs were achieved following immunization with phages screened with IMFS. However, no protection was elicited by those selected with NMFS. Conclusion The results show that the phagotopes are both antigenic and immunogenic, suggesting a potential use of phage displayed peptide as novel vaccines against S. japonicum.
文摘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.
文摘In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.
