1. Single Domain Antibody Screening Based on Phage Display Technology

The phage display technique was first developed by Smith et al. in 1985. The core of the technology is the fusion expression of the target gene in the phage coat protein gene by genetic engineering method, so that it can be displayed on the surface of the phage, then cloned and sequenced, and finally the DNA coding sequence of the target protein/polypeptide is obtained. This technology combines the genotype and functional phenotype (binding activity) with the help of the high amplification of bacteriophage. It is a very efficient screening system.

Since there is no problem of light and heavy chain matching, single-domain antibodies are more suitable for screening using phage display library technology. Phage single-domain antibody libraries are generally divided into three categories: immune libraries, natural libraries and fully synthetic libraries. Immune library refers to a single-domain antibody expression library constructed from pre-immunized animals (such as alpaca, etc.) by specifically enriching the target antigen-specific B cell genes and amplifying the heavy chain antibody VH gene to obtain a single domain antibody expression library. High affinity antibodies can be screened with a small storage capacity.

The limiting factor of the immune library is the characteristics of the immunogen. The strength of the humoral immune response and the epitope difference of different immunogens in animals may affect the quality and diversity of the final antibody obtained, and the homogenization of the antibody screened. The natural library is a single-domain antibody library constructed by amplifying all heavy chain antibody VH genes from B cells of animals that have not been immunized with the target antigen. The success rate of screening high-affinity single-domain antibodies from natural libraries is proportional to the capacity. In theory, as long as the capacity and diversity of the antibody library constructed is large enough (>10 CFU/m), it can be screened to target various High affinity single-domain antibodies against target antigens, even single-domain antibodies against highly conserved antigens (similar to self-antigens) in mammals.

Although the natural library has high diversity, due to the bias of the antibody gene family and the deletion of clones caused by innate immune tolerance, the diversity will be reduced due to the actual library construction operation. The synthetic single-domain antibody library constructed directly by gene synthesis method can further improve the diversity of the library by adjusting the framework region near the antigen-binding region in the antibody molecule, or it can be directly constructed based on the humanized VHH molecular framework.

2. Single-domain antibodies screening based on yeast display technology

Yeast belongs to eukaryotic single-celled organisms. The yeast used for antibody display technology is generally Saccharomyces cerevisiae. This yeast has a cell wall of about 200 nm thick. The budding yeast cell wall has a lectin protein on the surface, which can be matched with the relatively mating type. The Aga2p protein on yeast binds, and the single-domain antibody is expressed as a fusion protein with Aga2p protein and displayed on the surface of yeast cells.

The screening method of yeast display library generally uses magnetic bead-assisted cell sorting (MACS) and flow cytometry sorting (FACS). The main function of MACS is to reduce non-specific binding, usually on the basis of MACS screening.

Compared with phage display technology, yeast display technology has both advantages and disadvantages. On the one hand, yeast belongs to eukaryotic cells. The post-translational modification of antibodies is similar to mammalian cells. The stability of antibodies after glycosylation is high. At the same time, unknown conditions that may exist in mammalian expression systems are avoided. Secondly, the yeast display technology adopts the flow cytometric sorting method, and the controllability of the entire screening process is higher. On the other hand, the storage capacity of the yeast antibody library is generally smaller than that of the phage antibody library, up to 1x10, CFU, but with the optimization of technology, it has been reported that the capacity of yeast antibody library can be increased to 10CFU level, while the capacity of phage antibody library can be as high as 10CFU. Meanwhile, yeast display technology may be ineffective in screening oligomer antigens, because each yeast cell will display 1x10 to 1x10 antibody fragments, so one yeast cell may covalently bind multiple antigens at the same time, which may cause the affinity of the selected antibody to be less than expected.

3. Single-domain antibody screening based on bacterial display technology

In addition to the construction of phage libraries, Escherichia coli can also be used as a carrier for antibody display. E. coli is a Gram-negative bacterium. In addition to the bacterial inner membrane, there is a layer of bacterial outer membrane outside the cell wall. Antibody fragments are usually displayed on the inner membrane of E. coli bacteria, and the Fc segment of the antibody binds to lipoproteins on the membrane. This display and expression method is called Anchored periplasmic expression (APEx). However, due to E .coli bacterial cell walls and outer membranes exist, so the bacteria need to be transparently processed into the form of spheroplasts to bind to the antigen.

The technique of displaying antibodies using outer membranes has also been reported. For example, Salema and others display antibody fragments on Eha Aautotransporter or Intimin (tight adhesion protein). These two proteins are derived from E.coli 0157:H7( EHEC) the outer membrane of bacteria, and can be expressed and displayed in E. coli K-12 bacteria. Gram-positive bacteria Staphylococcus Staphylococcus carnosus can also be used for antibody display, this method is through the cell-wall anchoring domain of Staphylococcal protein A protein of Staphylococcus spp. transfers the antibody fragments from the cell membrane to the outside of the membrane. The screening method of bacterial display technology generally adopts flow Cell sorting (FACS), also combined with magnetic bead assisted cell sorting (MACS)

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