Method for analyzing epitope of protein

Abstract

The invention discloses a method for analyzing epitope of a protein, comprising the following steps of: (1) constructing a deoxyribonucleic acid (DNA) library of a target protein; inserting the DNA segment of the coding gene of the protein into a T carrier, wherein the T carrier contains the coding gene of yeast a lectin Aga2p; fusing a peptide coded by the DNA segment with the C end of the yeast a lectin Aga2p; (2) displaying the DNA library on yeast surfaces to obtain a yeast display library; (3) separating out yeasts combined with the antibody of the target protein; and (4) extracting plasmid sequencing and analyzing the epitope of the protein. The invention has the advantages that (1) yeast cells have multiple modification paths on the protein, can conveniently display the complicated high molecular weight protein and better simulates a space conformation; (2) the yeasts can be screened one by one by adopting flow cytometry (FACS) so as to improve the accuracy and the screening flux; (3) the yeasts can independently complete the self growth reproduction process, the operation is simple and convenient and the interference factors are few; and (4) the yeasts are not easy to propagate in the air or generate cross infection.

Description

technical field [0001] The present invention relates to a method for analyzing antigenic epitopes of proteins. Background technique [0002] The host's antibody response plays an important role in resisting pathogen infection, while the in vivo polyclonal antibody response consists of monoclonal antibodies directed against different antigens or different epitopes of the same antigen. An in-depth understanding of this complex process can provide key information for studying the pathogenesis of diseases, and can also provide an important theoretical basis for the development of efficient vaccines and disease treatment. [0003] Among the many research methods of antigenic epitopes, the phage display peptide library technology that emerged in 1990 (Scott, J.K.and G.P.Smith, Searching for peptide ligands with an epitope library. Science, 1990.249(4967): p.386-90. ), that is, use a randomly synthesized short peptide library (usually 6-12 amino acids, the library capacity can rea...

AI Technical Summary

Problems solved by technology

However, the phage display peptide library technology also has great limitations: (1) Most of the short peptide libraries that can be generated are linear epitopes, which cannot well mimic the conformational epitopes that account for more than 80% of antigenic epitopes; (2) The foreign molecules that can be accommodated by the capsid protein of phage are small and simple in structure, and lack of modification of foreign proteins. Many cytotoxic molecules and eukaryotic proteins in this system are difficult to express and display; (3) The individual phage is too small , mostly using affinity chromatography columns or affinity polystyrene plates for screening, the screening efficiency is low; (4) After each round of screening, the phage must re-infect the host cells in order to obtain enough virus particles for the next round of screening, recombinant virus The difference in infection ability between particles will result in certain amplification advantages; (5) phages are easily spread by air, thus easily causing cross-contamination

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