Abstract:
Abstract Phage display is a process by which a peptide or a protein is expressed as an exterior fusion to a surface protein of a phage particle. The peptide or protein sequence can be deduced from its encoding DNA sequence that resides in the phage particle or in a transductant. Amplification of the DNA of interest can take place by phage/transductant propagation or by polymerase chain reaction (PCR). By producing large populations of phage particles, each expressing a unique peptide or protein, peptide/protein libraries can be obtained. Peptides or proteins, interacting with defined molecular targets most often proteins can be isolated from such libraries by enrichment through repeated cycles of panning. Hence, phage display can be thought of as a ‘‘search engine’’ of protein^target interactions. The pioneering work of Smith (113) first demonstrated surface display of peptides in filamentous phage fd. This innovation was extended to peptide libraries of fd and M13 (19, 22, 108) and phagemid display was introduced (6). The display of proteins such as antibody domains and combinatorial antibody libraries soon followed (5, 59, 75). From the beginning of the 1990s phage-display-related publications have grown exponentially (128). Several reviews (some general, e.g., 58, 125; and many specialized) are available. There are numerous reports and several laboratory manuals (4) describing development and use of filamentous phage display in identification of peptide or protein interactions with simple organic compounds, antibodies, receptors, etc. Phage display is also a useful tool in protein engineering and directed evolution (44). Then there is the large sector of phage antibody display (64) and the more recent field of immune profiling and its implication for vaccine development (15, 112). Furthermore, complex targets such as cells (92) and whole tissues/organs (91) have been subjected to phage display analysis. These studies explore novel approaches for in vivo homing in gene/drug delivery (78), cancer surveillance/treatment (2, 86, 103), and imaging (127).Keywords:
Phagemid
Panning (audio)
Panning (audio)
Phagemid
Lytic cycle
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Panning (audio)
Phagemid
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Panning (audio)
Phagemid
Immunoglobulin Fab Fragments
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Panning (audio)
Phagemid
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Panning (audio)
Phagemid
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Abstract Bacteriophage display systems have been developed to allow the selection of a protein or peptide based on its ability to bind to a selected target molecule. The antibody‐display approach uses filamentous phage that express combinations of randomly assembled pairs of heavy and light chain genes either as a single chain antibody fragment or fragment antigen binding (Fab) format. These proteins are fused to the phage minor coat protein, pIII, to allow their expression and display on the surface of the phage, where they are available to bind antigen. Phage(s) expressing a particular sFv/Fab are enriched by panning on an immobilized target. Various antibody‐display phage libraries have been described and this system provides an alternative and powerful tool in generating specific antibodies.
Panning (audio)
Phagemid
Filamentous bacteriophage
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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 in vitro 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. This thesis lays the foundation for the theoretical and experimental research of bacteriophages.
Panning (audio)
Phagemid
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In this study,we constructed a phage display antibody library specific for gentamicin(GEN) to get the anti-getamicin single chain antibody(scFv).We used the anti-gentamicin hybridoma(2A3) as a source of gene to produce full-length scFv fragment.The full-length scFv fragment was ligated into the phagemid vector pCANTAB5E.Then the phagemid was transformed to E.coli TGl cells by electroporation.The size of antibody libraries was 6.5×106.The primary phage display library was riched by stabilized antigen immunoaffinity panning with GEN-OVA conjugates.9 clones congtained insert and showed GEN-positive in phage-ELISA,which lay the foundation for further immunological analysis.
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Phagemid
Insert (composites)
Biopanning
Single-chain variable fragment
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Objective Anti-activity platelet phage display library was constructed from mouse immunized with P-selectin, which has been used in clinical usage. Methods The heavy-chain and light-chain variable region gene(VH and VL)repertoire of immunoglobulin were amplified from the spleen cell mRNA by RT-PCR and joined by a DNA linker encoding peptide as a single-chain Fv(ScFv) fragment with overlap extension. These fragments were cloned into the phagemid pHEN1 and the phage display library was constructed. The affinity selection and ELISA were adopted for identification of specific phage antibody to P-selectin. Results After 5 rounds of panning, the high affinity ScFv gene was obtained, the sequence was confirmed with mouse antibody.Conclusion Phage display library of repertoire single chain antibody of anti P-selectin was successfully constructed, and gene of single chain antibody which have binding ability to P-selectin wass creened.
Panning (audio)
Phagemid
Single chain
Antibody Repertoire
Linker
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Objective:To obtain the gene sequence of antibody against human death receptor 5 by the phage surface display.Methods:By the technique of phage display,the heavy chain and light chain variable region genes(VH and VL) were assembled into single chain antibodies(scFv) with overlap extension reaction by L inker primer mix.The scFv were cloned into PAK100 phagemid and introduced into E.coli XL1-Blue.The phagemid containing bacterial colonies were infected with VSCM13,then the total surface display library of anti-human-death receptor 5 was established.The library was screened by death receptor 5 to get the antigen-specific phage clones.Positive clones were characterized by DNA sequencing.Results:After three panning rounds ofbinding-elution-amplification,10 individual clones were randomly selected and identified by enzyme-immunosorbent assay(ELISA),4 of which has the characteristic of DR5.Conclusion:The technique of phage display is a more creditable method to obtain antibody gene sequence.
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Phagemid
clone (Java method)
Single-chain variable fragment
Primer (cosmetics)
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