Application of Phage Display Technology
The phage display technology is to fuse the foreign DNA fragment encoding the polypeptide with the coding gene of the phage surface protein, and then present on the surface of the phage as a fusion protein, and the displayed polypeptide or protein can maintain relative spatial structure and biological activity, and display On the surface of the phage. A group of phage introduced with a variety of foreign genes constitutes a phage display library displaying a variety of exogenous peptides. This article is used to introduce some applications of phage display technology.
Antibody screening
The gene of the variable region of the antibody is inserted into the phage genome, and the expressed antibody is displayed on the surface of the phage, and a phage display antibody library is constructed, and the process of antibody production can be simulated in vitro to screen antibodies against any antigen. By screening antibodies against phage display antibody library technology relative to hybridoma technology, the cycle of antibody production can be shortened without immunization. It is also possible to screen antibodies which have weak immunogenic or toxic antigens in vivo and have a wide range of applications. The phage display antibody library technology is not limited by species, and antibody libraries of various species can be constructed. Antibodies screened from human natural libraries can be directly used for antibody drug research without humanization.
Discovery of new receptors and ligands
A random polypeptide sequence is displayed on the surface of the phage to obtain a phage display polypeptide library. The cells are used as screening targets, and differentially screened to obtain polypeptides that recognize specific cells. By studying the polypeptide sequence, a receptor protein specifically expressed on the cell surface can be further obtained. The 12-peptide library was screened by HCT116 cells, and a polypeptide which can specifically recognize colon cancer cells was selected from the library. Further analysis revealed that the polypeptide specifically recognizes a-enolase. This protein is expected to be a target for the treatment of colon cancer and to screen for the treatment of colon cancer. The obtained polypeptide sequence can also be used as a carrier for anticancer drugs.
Protein interaction study
Protein interactions are indispensable in life processes, and phage displayed peptide libraries are composed of random short peptide sequences of specific length. By affinity-panning the random library with a target protein (such as a receptor), a short peptide sequence can be obtained. The obtained sequences were sequenced and analyzed, and the corresponding short peptides were synthesized, so that the interaction between the two proteins can be studied. A number of important macromolecules such as growth hormone receptors, insulin receptors, insulin-like growth factor receptors, and agonists and elixirs of TNF-a receptors have been successfully identified by this method.
Epitope analysis
The antibody is used as a screening protein, and a phage which can specifically bind to the antibody is selected from a random polypeptide library displayed by the phage, and the epitope recognized by the antibody is obtained by sequencing analysis. The technology provides a basis for antigen-antibody reaction mechanism research, diagnostic reagent development, vaccine preparation and the like.
The current epitope identification technology can achieve:
1. Preparation of monoclonal antibody and diagnostic monoclonal antibody;
2. Development of therapeutic and prophylactic recombinant polyvalent peptide vaccines including “general” targets;
3. Development of single epitope or recombinant multi-epitope peptide detection antigen;
4. Screening for new specific diagnostic markers such as diseases and tumors based on epitope motifs;
5. High-throughput found all conserved and specific epitopes in homologous proteins;
6. Screening functional antibody epitopes or antibody neutralizing and accessibility epitopes;
7. Analysis of viral genetic evolution and variation at the epitope level provides direct evidence of antigenic drift and metastasis.
Humanized transformation of antibodies
The proportion of human monoclonal antibody is increasing, and the target of monoclonal antibody is gradually diversified. In addition to the traditional cell surface antigen, it also includes common cytokines. Some monoclonal antibodies can even recognize multiple epitopes. And the structure of the monoclonal antibody is not limited to the intact monoclonal antibody molecule. The treatment options for joint small molecules and so on have gradually increased, and are increasingly valued by medical workers. Therefore, as a high-tech drug, the technological level of the monoclonal antibody drug company determines its competitiveness, and also determines the therapeutic effect and market value of the drug.
Bispecific antibody (BsAb) preparation
By combining two antibody fragments targeting different antigens by genetic engineering, there are two antigen binding sites, which can exert synergistic effects and thereby improve the therapeutic effect. However, there are many types of bispecific antibodies, and the selection is based on the final application.
Enzyme inhibitor screening
β-ketoacyl-ACP reductase is a highly conserved and widely-existing enzyme in the biosynthesis and metabolism of prokaryotic fatty acids. This protein is used as a target protein for screening, and the inhibitor of the enzyme is screened from the phage peptide library. A new type of antibacterial agent. A series of highly efficient insecticides and herbicides have been developed and developed for target enzymes such as acetylcholinesterase, trehalase, acetolactate synthase, acetyl CoA carboxylase and glutamine synthetase.
Directional transformation of protein
Protein-directed transformation refers to the mutation of a specific coding sequence of a protein or domain by means of cassette mutation, error-prone PCR, etc., and a mutant library producing a protein or a domain is presented on the surface of the phage, obtained by affinity screening. Phage clones that have been altered in orientation, their primary structure can be deduced from the sequence of DNA, and can be used to screen for cytokines with stronger receptor binding ability, new enzyme inhibitors, DNA binding new sites for transcription factors, New cytokine antagonists, novel enzymes, and proteins that enhance biological activity.
After nearly 20 years of development and improvement, phage display technology has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, epitope research and cell signal transduction research. The phage display system mimics the natural immune system, making it possible to model the in vivo antibody production process and build a high-affinity antibody library.