Phage Display Library Construction Service

Alpha Lifetech has been deeply engaged in Phage display technology for many years. It has built a perfect stable Phage display technology platform that saves time for scientific research time or project research and facilitates subsequent production. Alpha Lifetech can also provide customers with services such as vhh antibody production, scFv antibody production, Fab Antibody production.

Alpha Lifetech has a comprehensive M13/T7 phage display platform to ensure the construction and production of antibody libraries. We can also provide customized services, such as antibody scFv production and high-throughput antibody screening, to meet your needs.

Introduction to Phage display

Phage display technology is a technique that uses phages (a virus that infects bacteria) to search for functional binding molecules of specific proteins or peptides. Phage antibody library construction techniques include Fab antibody library construction, antibody scFv library construction, nanobody library construction, etc. According to the types of bacteriophages, they can be divided into M13, T7, T4, λ, and other systems. According to the library type, it can be divided into random peptide library, cDNA library, antibody library, and protein library. Phage display technology is simple to operate and inexpensive to use. However, this technique limits the diversity of molecular genetics in the library that can't express too long sequences,

At present, phage display technology is widely used. Among them, there are significant achievements in the research and development of new vaccines (low-cost and efficient synthetic vaccines), the development of antibody drugs (screening enzyme inhibitors), cell signal transduction (screening of simulated epitopes), and the research of antigen epitopes (preparation of monoclonal antibodies).

Introduction to T7 Bacteriophage

T7 bacteriophage is double-stranded DNA, the length of the DNA is 40kb and is wrapped in a capsid with a diameter of 60nm. The connector between the head and tail is a ring structure composed of multiple copies of gp8. The head and core of the t7 bacteriophage form a cylindrical structure that binds to gp8 and can connect the head and tail.

Fig. 1 Schematic representation of the T7 bacteriophage. (Reference: Advances in the T7 phage display system (Review))

Introduction to M13 Bacteriophage

The bacteriophage M13 belongs to a group of filamentous phages collectively called Ff phages. It has a length of 900 nm and a width of 6.5 nm. It contains a genome of single-stranded DNA (ssDNA) with a length of 6407 bp that consists of nine genes encoding 11 different proteins. Among them, the five proteins are coat proteins, and the six proteins are involved in the replication and assembly of the phage. The highest concentration of coat proteins is the capsid protein G8P, which is made up of about 2,700 protein units and forms an envelope around the chromosome.

Fig. 2 Schematic representation of the bacteriophage M13. (Reference: Basics of Antibody Phage Display Technology)

Introduction to Phage Display Antibody Library

Antibody discovery has become increasingly important in modern medicine. Different antibody discovery approaches exist, but the phage display antibody library is used more in medical science.

Since 1990, different antibody formats have been used to construct phage libraries, including VHs, VHHs, scFvs, diabodies, and Fab antibodies. The scFv composed of VH and VL structural domains are single-chain antibodies used to construct antibody scFv libraries. scFv is characterized by a short half-life and low immunogenicity. Fab Antibody Library consists of VH, VL, CL, and CH1, which can rapidly screen out ideal antibodies with high affinity. The smallest unit that can bind the target antigen-VHH constitutes the nanobody library currently, VHH has the advantages of simple structure, small volume, high solubility, good stability, and easy preparation and expression. Synthetic nanobody (Nb) libraries are emerging as an attractive alternative to animal immunization for the stable, high-affinity synthetic nanobody. Generally, these antibody fragments are fused to the G3P of the M13 phage, and by cloning large numbers of genes encoding an antibody fragment, large phage display antibody libraries can be generated from which many diverse antibodies can be selected.

Phage Library Construction Process

The process of phage library construction is as follows: specific primers are designed for PCR amplification that obtains the products, which are enzyme-ligated with the T7/M13 phage vector, and the recombinant phage plasmid is successfully constructed. The recombinant phage plasmid was transformed into TG1 receptor cells, then coated on a medium containing appropriate antibiotics, and expanded culture was performed after screening the transformers. After multiple rounds of culture, the phage replicates replication times in the bacteria and successfully expresses the target protein or polypeptide. Then the phage library is purified to remove some impurities and unbound phages.

Variable regions (VH and VL) correlated with antibody diversity, and VH and VL were inserted into the phage vector along with a sequence encoding the phage protein PIII. After assembly, the phage particle is exposed and fused with the N-terminal of the minor coat protein III to form a functional antibody fragment, obtaining a library containing the antibody DNA sequence.

After the end of immunization, the titer was detected, and animal blood was taken after the titer was qualified. Lymphocytes are isolated from the blood, RNA is extracted, RT-PCR amplifies the target fragment, and the V region gene fragment is obtained. The V gene is amplified using a specific primer.

The natural libraries created include poor immunogenic antibodies in animals that can target any target. Phage antibodies that bind specifically to the antigen can be collected using screening techniques that fix or label the antigen.

The target antigen is fixed to a solid carrier, such as a microplate hole, or coupled to a magnetic bead. The phage antibody library is then added to bind it to the antigen. After multiple elutions, the low-affinity or non-specific phages are washed away and those displaying specific antibodies are retained.

Application of Phage Display

*Antibody discovery has become increasingly important in modern medicine. Different antibody discovery approaches exist, but phage display technology is used more in medical science. Since 1990, different antibody formats have been used to construct phage libraries, including VHs, VHHs, scFvs, diabodies, and Fab antibodies.
*Phage peptide libraries allow the rapid determination of the sequence of protein epitopes and have become a powerful tool for investigating the interaction between epitopes and antigen receptors
*Antibody fragments are fused to the G3P of the M13 phage, and by cloning large numbers of genes encoding an antibody fragment, large phage display antibody libraries can be generated from which many diverse antibodies can be selected.
*The T7 phage display system has many advantages, including simplicity, high safety, stability, easy storage, and transportation, so the system is used in preventive and therapeutic vaccines.
*The T7 phage display system can detect various antigens, such as surface antigens of pathogenic microorganisms and cancer antigens.

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