Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—...Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—suchas animal immunization or B cell isolation from infected individuals—are often hindered by biosafety concerns,lengthy development timelines,and limited adaptability during outbreaks.In the present study,we aimed toestablish a robust and rapid in vitro platform for the efficient isolation of neutralizing antibodies targetingconserved viral epitopes.We developed an epitope-guided negative screening strategy that integrates phagedisplay technology with rational antigen mutagenesis to exclude antibodies against variable regions whileenriching for those that recognize functionally constrained epitopes.When applied to the receptor-binding domainof severe acute respiratory syndrome coronavirus 2,this method enabled the identification of six neutralizingantibodies(one IgG and five nanobodies)exhibiting broad-spectrum neutralizing activity across multiple viralvariants.Notably,antibodies recognizing distinct epitopes demonstrated significant synergistic neutralizationwhen used in combination(P<0.05).This screening approach facilitates the rapid discovery of potent andmutation-resistant antibodies and holds promise for application to other emerging pathogens.Our findingsunderscore the potential of epitope-guided,in vitro platforms in expediting therapeutic antibody developmentunder conditions of high biosafety requirements.展开更多
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 t...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.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 epito...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
The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD...The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.展开更多
AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search f...AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.展开更多
The blood brain barrier represents a formidable obstacle for the transport of most systemati- cally administered neurodiagnostics and neurotherapeutics to the brain. Phage display is a high throughput screening strate...The blood brain barrier represents a formidable obstacle for the transport of most systemati- cally administered neurodiagnostics and neurotherapeutics to the brain. Phage display is a high throughput screening strategy that can be used for the construction of nanomaterial peptide libraries. These libraries can be screened for finding brain targeting peptide ligands. Surface functionalization of a variety of nanocarriers with these brain homing peptides is a sophisticated way to develop nanobiotechnology-based drug delivery platforms that are able to cross the blood brain barrier. These efficient drug delivery systems raise our hopes for the diagnosis and treatment of various brain disorders in the future.展开更多
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.Li...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.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
Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red ...Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.展开更多
Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20<sup>th</sup> century has reduced significantly the rate of the viral infection. However, current...Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20<sup>th</sup> century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.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
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 ...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.展开更多
To make further investigation of the IgE antibodyrepertoire in Trichosanthin (TCS) allergic responses, amurine IgE phage surface display library was constructed(3.0×105 independent clones). We first constructed t...To make further investigation of the IgE antibodyrepertoire in Trichosanthin (TCS) allergic responses, amurine IgE phage surface display library was constructed(3.0×105 independent clones). We first constructed theVe cDNA library (4.6×105 independent clones) and VκcDNA library (3.0×105 independent clones). Then, theVε and Vκ gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs wasthus constructed. The diversity of Vε from this library wasanalyzed and proved. Fab clones with high specificity toTCS have been screened out.展开更多
Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the targ...Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the target is obtained. Antibody phage display has become the first and most widely used <i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> screening technology. Phage display derivatives play an important role in the diagnosis and treatment of diseases. This article reviews the phage display system of phage display technology, the size and classification of antibody libraries and their applications, and discusses the application prospects and challenges of phage display technology.</span><span style="font-family:Verdana;"> </span><span style="font-family:Verdana;">This thesis lays the foundation for the theoretical and experimental research of bacteriophages.</span>展开更多
Molecular imaging plays important roles in many fields,including disease diagnosis,therapeutic efficacy evaluation,intraoperative imaging guidance,drug metabolism monitoring,and patient selection for appropriate treat...Molecular imaging plays important roles in many fields,including disease diagnosis,therapeutic efficacy evaluation,intraoperative imaging guidance,drug metabolism monitoring,and patient selection for appropriate treatment.As a key component,the targeting ligand determines the specificity,affinity,and in vivo performance of molecular imaging probes.In this review,highthroughput screening and biological display platforms for the discovery of ligands applicable to molecular imaging are briefly reviewed.Basic information on ligand development for molecular imaging is first introduced,followed by a presentation of various selection platforms and typical or iterative cases.The features,advantages,limitations,and application scope of screening and display platforms are compared and discussed.Last,a basic selection strategy and a perspective for protein-based ligands are provided.展开更多
The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin ...The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.展开更多
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...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.展开更多
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 c...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.展开更多
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 （T...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.展开更多
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. Brea...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.展开更多
基金supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(Grant No.22KJB310001 to W.G.)the Special Project of the Jiangsu Provincial Department of Science and Technology(Grant No.BE2023603 to W.G.)the Nanjing Medical University Science and Technology Development Foundation(Grant No.NMUB20210006 to W.G.).
文摘Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—suchas animal immunization or B cell isolation from infected individuals—are often hindered by biosafety concerns,lengthy development timelines,and limited adaptability during outbreaks.In the present study,we aimed toestablish a robust and rapid in vitro platform for the efficient isolation of neutralizing antibodies targetingconserved viral epitopes.We developed an epitope-guided negative screening strategy that integrates phagedisplay technology with rational antigen mutagenesis to exclude antibodies against variable regions whileenriching for those that recognize functionally constrained epitopes.When applied to the receptor-binding domainof severe acute respiratory syndrome coronavirus 2,this method enabled the identification of six neutralizingantibodies(one IgG and five nanobodies)exhibiting broad-spectrum neutralizing activity across multiple viralvariants.Notably,antibodies recognizing distinct epitopes demonstrated significant synergistic neutralizationwhen used in combination(P<0.05).This screening approach facilitates the rapid discovery of potent andmutation-resistant antibodies and holds promise for application to other emerging pathogens.Our findingsunderscore the potential of epitope-guided,in vitro platforms in expediting therapeutic antibody developmentunder conditions of high biosafety requirements.
基金This work was supported by grants from the National Natural Science Foundation of China(No.30400111)the Natural Science Foundation of Jiangsu Province(No.BK2004041).
文摘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.
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.
基金supported by research grants from the National Natural Science Foundation of China(No.30271l87)the Shanghai Science and Technology Development Foundation(No.02DJ14015)
文摘The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.
基金Supported by the Major State Basic Research Development Program of China, 973 Program, No. 2002CB513100
文摘AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
文摘The blood brain barrier represents a formidable obstacle for the transport of most systemati- cally administered neurodiagnostics and neurotherapeutics to the brain. Phage display is a high throughput screening strategy that can be used for the construction of nanomaterial peptide libraries. These libraries can be screened for finding brain targeting peptide ligands. Surface functionalization of a variety of nanocarriers with these brain homing peptides is a sophisticated way to develop nanobiotechnology-based drug delivery platforms that are able to cross the blood brain barrier. These efficient drug delivery systems raise our hopes for the diagnosis and treatment of various brain disorders in the future.
基金supported by the National Natural Science Foundation of China(31941001 and 32002292)the Major Science and Technology Project of Henan Province,China(221100110600)the Natural Science Foundation of Henan Province(202300410199).
文摘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.
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 National Natural Science Foundation of China(Nos.31971561,31370217,31701724)the Research Program of Jiangsu Province of China。
文摘Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.
文摘Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20<sup>th</sup> century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.
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This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30200295).
文摘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.
文摘To make further investigation of the IgE antibodyrepertoire in Trichosanthin (TCS) allergic responses, amurine IgE phage surface display library was constructed(3.0×105 independent clones). We first constructed theVe cDNA library (4.6×105 independent clones) and VκcDNA library (3.0×105 independent clones). Then, theVε and Vκ gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs wasthus constructed. The diversity of Vε from this library wasanalyzed and proved. Fab clones with high specificity toTCS have been screened out.
文摘Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the target is obtained. Antibody phage display has become the first and most widely used <i><span style="font-family:Verdana;">in vitro</span></i><span style="font-family:Verdana;"> screening technology. Phage display derivatives play an important role in the diagnosis and treatment of diseases. This article reviews the phage display system of phage display technology, the size and classification of antibody libraries and their applications, and discusses the application prospects and challenges of phage display technology.</span><span style="font-family:Verdana;"> </span><span style="font-family:Verdana;">This thesis lays the foundation for the theoretical and experimental research of bacteriophages.</span>
基金Key Laboratory of Bioresource Research and Development of Liaoning Province,Grant/Award Number:2022JH13/10200026National Natural Science Foundation of China,Grant/Award Number:U2267221。
文摘Molecular imaging plays important roles in many fields,including disease diagnosis,therapeutic efficacy evaluation,intraoperative imaging guidance,drug metabolism monitoring,and patient selection for appropriate treatment.As a key component,the targeting ligand determines the specificity,affinity,and in vivo performance of molecular imaging probes.In this review,highthroughput screening and biological display platforms for the discovery of ligands applicable to molecular imaging are briefly reviewed.Basic information on ligand development for molecular imaging is first introduced,followed by a presentation of various selection platforms and typical or iterative cases.The features,advantages,limitations,and application scope of screening and display platforms are compared and discussed.Last,a basic selection strategy and a perspective for protein-based ligands are provided.
基金The Science and Technology Project Foundation of Guangzhou,China(Grant No.201604020106)Natural Science Foundation of Guangdong Province(Grant No.1814050002837)
文摘The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.
文摘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.
基金Supported by the National Natural Science Foundation of China(Nos 20072010 and 20572035) and the Science Foundation ofJilin University(Nos419070100087 and 01208006)
文摘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.
文摘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.
基金Supported by a grant from Science and Technology Planning Project ofGuangdong Province(No:2010B031600066)
文摘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.
