Single-chain Antibody Library Construction Technology Introduction-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Single-chain Antibody Library Construction Technology Introduction

Recent advances in antibody phage display technology have made it possible to overcome these limitations and generate human monoclonal antibodies that recognize any desired antigen. For phage display, the antigen binding regions of the VH and VL genes are cloned and used to construct scFv/Fab gene libraries. Phage antibody libraries are generated by cloning these antibody libraries as fusion proteins with a minor coat protein (pIII protein) of the phage. Each phage has a functional antibody protein on its surface and contains the gene encoding the antibody, which is integrated into the phage genome. Specific phage antibodies that specifically bind to proteins and small molecules can be separated from non-binding phage antibodies using affinity chromatography. This strategy does not require immunization, antibody gene cloning, and antibody fragments are well expressed in E. coli. The number and affinity of antibodies generated against a specific antigen are a function of library size and diversity. The larger the library, the greater the number of high-affinity antibodies generated.

I. scFv Library Construction

New Zealand white rabbits were immunized (no less than 5 injections), and the antibody titer (>10^5) was tested by ELISA. If the titer was qualified, blood was collected to isolate PBMC. The VH gene library was PCR amplified from the pCITE-VH library using 300 ng of library plasmid DNA as a template, Vent DNA polymerase, CITE3 primers, and an equimolar mixture of HuJH primers. The VL gene for scFv assembly was obtained from a previously constructed scFv phage antibody library. The VL gene library, including the DNA encoding the scFv peptide linker, was amplified from 300 ng of library plasmid DNA using an equal mixture of Vent DNA polymerase, Gene3 primers, and RHuJH primers. The amplified VH and VL genes were agarose gel purified and spliced together using overlap extension PCR to form the scFv gene library. The schematic diagram is shown below:

Schematic diagram of library construction method-Tekbiotech.png

Figure 1 Schematic diagram of library construction method

Note: (A) VH and linker-VL gene libraries were PCR-derived from plasmid DNA from different libraries. VH genes were amplified using plasmid-specific primers and an equal mixture of HuJH primers. Linker DNA and VL genes were amplified using plasmid-specific primers and equimolar RHuJH primers. RHuJH primers are complementary to HuJH primers; (B) VH and linker DNA-VL gene libraries were aggregated into scFv gene libraries; (C) The assembled scFv gene library was cut with restriction endonucleases NcoI and NotI and cloned into plasmid pHEN1 for phage display.

II. Cloning of ScFv Gene Library

The purified DNA of the scFv gene library was double-digested, and the digested products were ligated to the vector digested with the same enzymes. The ligated products were purified and electroporated into Escherichia coli TG1. The cells were cultured in SOC for 1 h and then cultured on TYE-AG medium (PharmaciaBiotech) N plates. All ligated samples were electroporated more than 10 times, and their conversion efficiency was calculated through a series of dilutions. Scrape the colonies from the 5th plate, resuspend them in YT-AG containing 15% (v/v) glycerol, and store the library at -70°C.

III. scFv Purification and Affinity Determination

The scFv gene needs to be subcloned, expressed, and purified to achieve homogeneity. Recombinant DNA and gene amplification technologies make it possible to clone antibody genes in bacteria. This "non-death" of antibody genes makes it technically feasible to quickly produce antibodies (or antibody fragments) in bacterial culture and genetically manipulate their structure, so that antibodies against the Y antigen can eventually be produced, and even smaller antibodies with higher affinity and higher specificity can be designed. The scFv dissociation equilibrium constant (Kd) is calculated from the association (kon) and dissociation (koff) rate constants measured in BIAcore using surface plasmon resonance.

Recombinant phage antibody libraries constructed using variable region genes of immune or non-immune animal or human lymphocyte antibodies are a powerful way to obtain recombinant phage antibodies by displaying functional antibody fragments on the phage surface and screening fusion phages with antigen binding activity. The usefulness of the phage system is that the essence of the natural antibody system can be replicated in the phage through connection recognition and replication.

TekBiotech has been committed to antibody research for many years. We have accumulated rich experience in antibody discovery and can provide customers with single-chain Fv (scFv) antibody library construction services based on antibody phage display library technology. Single-chain antibodies (scFv) have the advantages of small molecular weight, strong tissue penetration, and easy genetic engineering. TekBiotech uses antibody phage display library technology to prepare high-affinity and structurally stable scFv antibodies.

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