Because of the advantages of easy preparation, easy modification, and low immunogenicity, the importance of peptides in drug screening and treatment has been increasingly valued by researchers. Peptides are divided into linear peptides and cyclic peptides according to the structure of peptide bonds. Among them, linear peptides are the most widely and deeply studied. The genes inserted into the commonly used phage random peptide library are often displayed on the phage surface in the form of linear linear peptides after phage expression. However, linear peptides are easily degraded rapidly under the action of various enzymes after entering the human body, while the adjusted cyclic peptides will form a fixed conformation under the action of disulfide bonds or other binding bonds, which will not only greatly improve the metabolic stability and bioavailability, but also better meet the binding requirements of receptor-ligand (antibody-antigen) conformation.

一．Classification of Peptide Libraries

There are many types of constructed phage peptide libraries: according to different expression systems, they are divided into phage-derived vector expression peptide libraries and phagemid vector expression peptide libraries; according to the position of the fusion of the polypeptide and the coat protein, they are divided into gpI phage peptide libraries, gpV cyclic phage peptide libraries, etc.; according to the length of the polypeptide, they are divided into 6-peptide libraries, 8-peptide libraries, 15-peptide libraries, etc.; according to the different valences of the polypeptide, they are divided into monovalent phage peptide libraries and multivalent phage peptide libraries; according to the spatial conformation of the polypeptide, they are divided into linear phage peptide libraries and conformational phage peptide libraries; according to the size of the diversity of polypeptide sequences contained in the peptide library, they are divided into partial random peptide libraries and random peptide libraries.

Current research has the most extensive application of random heptapeptide, dodecapeptide and cyclic heptapeptide libraries. Random linear heptapeptide and dodecapeptide peptide libraries have simple structures and are suitable for the screening of mimetic epitopes. Random cyclic heptapeptide libraries have a ring structure and can be used for binding domain screening.

二．Method for Constructing Peptide Library

Biological gold panning method: the target molecule is coated on the solid phase medium, the phage peptide library is added to adsorb it, the non-affinity or low-affinity phages are washed away, and the equal-affinity phages are recovered. After several rounds of "panning", specific phage peptides can be enriched. The target protein used for phage screening can be directly adsorbed on the ELISA plate, or fixed on biological small magnetic beads, or the biotinylated target molecule can be fixed on the ELISA wells or biological magnetic beads coated with streptavidin (using the high affinity of streptavidin and biotin).

Selection of infectious phage (SIP): Replace the P protein with a specific peptide or protein to make the phage lose its infectivity, and then restore the infectivity through the binding of these peptides or proteins to their ligands. This method directly links the interaction between protein ligands and the infection amplification of phages, which can enable specific screening and phage amplification to be carried out simultaneously.

Delayed infectivity screening: Utilize the versatility of bacterial outer membrane protein A in the bacterial surface display system to fuse the coding sequence of the target protein to the hybrid outer membrane protein A for display. When the phage is captured or panned, the bacteria are transferred to 37°C for culture. The phage regains the ability to infect due to the expression of F cilia and becomes the selected clone. This method is the opposite of the conventional screening process, that is, only the selected bacteria can be infected by the captured phage, so it is also called "reverse screening".

三．Construction Process of Peptide Library (taking M13 phage random peptide library as an example)

1. Construction of M13 Random Peptide Library and Library Capacity Test

10μL of ligation product is electrotransformed into 100μL pre-cooled electrotransformation competent bacteria. After the transformed bacteria are added with 2mL SOC culture medium, cultured at 37°C for 1h, 1μL is taken for gradient dilution, and coated on a plate containing 50 μg/mL ampicillin to check the library capacity of the random peptide library. The remaining SOC cultures were added with 100 mL of SB culture medium containing 50 μg/mL ampicillin and 10 μg/mL tetracycline. After culturing at 37°C for 2 hours, 10^12 plaque-forming units (pfu) of helper phage VSC M13 were added. The culture was continued for 2 hours. Centrifuged at 5000 rpm for 15 min. After discarding the supernatant, 100 ml of SB culture medium containing 50 μg/mL ampicillin and 10 μg/mL tetracycline was added again. Cultured at 37°C overnight. The next day, centrifuged at 5000 rpm for 15 min to collect the supernatant. PEG8000 was added to 4%. NaCl was added to 3%, and after ice bathing for 30 minutes, the supernatant was discarded after centrifugation at 9000 rρm. The precipitate was re-dissolved with 2 ml of PBS solution containing 1% BSA, centrifuged at 10000 rpm for 5 min, the insoluble matter was discarded, and the supernatant was collected to be the filamentous phage random peptide library.

2. Randomness Test of M13 Phage Random Peptide Library

Take 1μL M13 phage random peptide library and dilute it to a titer of about 100 plaque forming units (pfu) according to a certain ratio. Take five dilutions and add 100μL F' XL1-blue bacterial solution with OD=1.0 respectively. Leave it at room temperature for 15 min, spread it on 5 ampicillin culture plates, and culture it at 37°C overnight. The next day, calculate the plaque forming units (pfu), pick a well-separated plaque on each ampicillin culture plate for amplification and culture, extract the single-stranded template to determine the exogenous sequence, and analyze the randomness of the peptide library.

四．Application of Phage Peptide Library Technology

1. Gene therapy: Compared with the gene therapy vectors currently used, phages have obvious advantages: simple and cheap preparation; no phagocytosis to mammalian cells; relatively simple genetic modification and construction.

2. Antigenic epitope research: Antigenic epitope is the active center where antigen and antibody combine. The study of specific antigen active center is conducive to revealing the mechanism of antigen-antibody interaction. For spatial conformational antigenic determinants, the corresponding antibodies can be used to separate the simulated antigenic epitopes from the peptide library.

3. Determine nucleic acid binding proteins: For specific DNA or RNA, constructing a random library of DNA or RNA binding domains for screening can determine its specific binding protein.

4. Gene vaccine research: Screen simulated epitopes that bind to antibodies and have strong immunogenicity from the epitope library, then immunize mice with simulated peptides or phage peptides and detect the mice and the antibody levels produced by the mice to determine the prospects of the screened phage peptides as vaccines.

5. Drug design and drug development: Using the full sequence of receptors or cytokines or a certain extracellular segment as the target molecule, screen active (activation or antagonism) phage peptides from the peptide library, and synthesize active peptides after sequencing. These small peptide sequences can be used as the basis for the design of small molecule peptide drugs.

The phage display platform provided by Tek Biotech is an ultra-high throughput ligand screening method. Through this technical platform, up to 10^13/mL of phage-displayed peptide or antibody clones can be constructed, and the average number of independent clones can be 10^9-10^10/mL. After subsequent rounds of screening, up to 10^5 effective peptide or antibody sequences can be provided, providing a solid research foundation for subsequent enzyme substrates, protein ligands and high-specificity antibody searches. At the same time, in conjunction with Tek Biotech's affinity screening platform, the affinity ranking of different peptides or antibodies with target protein targets can be obtained in a short time (2-3 days). Based on the phage platform, TechBio can construct random 7-peptide libraries, 12-peptide libraries, 15-peptide libraries, etc. for customers, which can fully meet the different needs of customers.