Phage display technology is a powerful gene expression screening technology, which was first described by SmithMl et al. in Science in 1985. The basic principle is to clone the gene of the foreign protein into the genomic DNA of the phage, thereby expressing the specific foreign protein on the surface of the phage. Ellis SEp et al. pointed out that using phage display peptide library can screen and determine the antigen of nematode vaccine, which is a new method of vaccine antigen identification. In recent years, the increase in diseases caused by epidemic viruses makes antiviral polypeptides a very promising method for prevention and treatment. Castel G et al. pointed out that recombinant polypeptides specifically expressed by phage display can be applied to antiviral research and drug development.
Based on phage display and PCR cloning technology, British scientists Winter et al. first published an article in Nature to describe the phage antibody library technology (https://www.creative-biolabs.com/phage-display-service.html), which is to recombine antibody heavy and light chain variable region genes with phage coat protein genes, and display antibody fragments Fab or scFv and phage coat protein in the form of fusion proteins on the surface of phage particles for rapid, efficient screening and enrichment of specific antibodies against a certain antigen. It has fundamentally changed the traditional monoclonal antibody preparation process, in which way fully human antibodies can be obtained, with unique advantages in the diagnosis and treatment of HIV and other viral infections and tumors.
Basic principles and procedures
The principle of phage antibody library technology is to use PCR technology to amplify a complete set of antibody heavy chain variable region (VH) and light chain variable region (VL) genes from human immune cells, and clone them into a phage vector, which is then expressed on the surface of its shell in the form of a fusion protein. In this way, the presence of antibody genes in the phage DNA and the expression of antibody molecules on the surface of the phage DNA contribute to using antigen-antibody specific binding to screen out the required antibodies for clonal amplification. By expressing the antibody gene in a secreted manner, soluble antibody fragments can be obtained. In library construction, if VH and VL are randomly combined, a combinatorial antibody library can be established. If the antibody mRNA is from an unimmunized person, a human natural antibody library can be established without cell fusion.
Phage display antibody library construction usually includes the following processes:
1. Isolate B lymphocytes from peripheral blood, spleen, lymph nodes, and other tissues. Extract mRNA for reverse transcription into cDNA.
2. Use antibody light chain and heavy chain primers to amplify different Ig gene fragments by PCR technology.
3. Construct phage vector. As of phage, filamentous phage, and phagemid, the latter two are commonly used vectors for constructing surface display antibody library.
4. The expression vector transforms bacteria to construct a full set of antibody libraries. Through multiple rounds of antigen affinity adsorption, elution, and amplification, antigen-specific antibody clones are finally screened out.
Features of phage antibody library technology
* Simulate a full set of natural antibody libraries.
The antibody library can reach or exceed 1011 storage capacity to cover all clones of B cells. The exogenous genes for library construction come from mmDNA extracted from human peripheral blood, bone marrow, or lymphocytes of the spleen, which is reversely transcribed to form cDNA. These mRNAs are the total mRNA of human polyclonal cells. The primers used are collected from multiple human bodies and are universal in human species. The random recombination of the antibody's VH and VL genes also increases the antibody diversity.
* Avoid using artificial immunity and hybridoma technology.
Due to the large capacity and extremely high screening efficiency of the antibody library, it is possible to utilize any antibody gene to prepare antibodies by genetic engineering methods, thereby avoiding the use of artificial immune animals and cell fusion technology.
* Humanized antibodies with high affinity can be obtained.
In the phage antibody library technology, the random recombination of VH and VL genes mimics the process of in vivo antibody affinity maturation. The antibody genes used are from the human body. Therefore, the products are high-affinity humanized antibodies.
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