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Construction and Evaluation of Single-chain Antibody Library

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Abstract: As a key genetically engineered antibody, single-chain antibody consists of three parts, the antibody light chain variable region, the heavy chain variable region and a segment of the bridge polypeptide chain. The single-chain antibody not only has strong targeting, but also is not easy to dissociate, has a simple structure, is easy to operate and transform, and is advantageous for antibody library screening. At present, there are two main ways to construct single-chain antibodies: 1. Amplification of antibody light chain and heavy chain variable regions from hybridism cell lines, but the construction of single-chain antibodies has a rejection reaction. 2. RNA was extracted from canine spleen B lymphocytes, reverse transcribed into cDNA, and antibody variable region was obtained. The phage display antibody library technology is a new technology currently available for the preparation of human monoclonal antibodies. It uses the RT-PCR method to amplify the human immunoglobulin full set variable region gene and then recombine into the prokaryotic expression vector, and forms a fusion protein with the phage coat protein to express the antibody fragment on the surface of the phage, and screens by panning. The specific variable region gene can conveniently and efficiently perform high-throughput screening of antibodies, and opens up a simple and rapid production route for the preparation of human monoclonal antibodies. A key aspect of this technology is the construction of a library of human phage antibodies.

Keywords: antibody library, single-chain antibody, construction

Phage single-chain antibody library for pancreatic cancer

Pancreatic cancer is one of the most malignant tumors known to date. Pancreatic cancer is the fourth leading cause of cancer death in Europe and America due to the insidious pancreatic cancer and lack of specific symptoms and signs. The phage antibody library technology has opened up new fields for the research of genetically engineered antibodies, and has been used in the diagnosis and prevention of infectious diseases, the identification of autoimmune diseases and viral diseases, imaging analysis of tumors and targeted therapy or gene therapy. The field shows great potential and broad application prospects. Compared with the traditional hybridism technology, the technology is simple, easy to produce, low in production cost, large in screening capacity, and can be prepared in large quantities by fermentation.

The total RNA of peripheral lymphocytes of patients was extracted, and the H chain and L chain variable region genes were amplified by RT-PCR. The VL fragments were randomly spliced into ScFv fragments by SOE-PCR. The ScFv fragment was then cloned into a specific vector and electroporated into a competent strain, rescued by the helper phage M13K07, and a human phage single-chain antibody library of pancreatic cancer was obtained, which aims to lay a foundation for the biological treatment of pancreatic cancer.

Pneumoconiosis phage single-chain antibody library

At present, pneumoconiosis is still the most common type of occupational disease in China with the highest incidence rate, the risk of death, and the most serious harm to workers. So far, there is no cure for it, which can only be eliminated or reversed according to the condition. Delaying the progression of the disease, therefore, active prevention and early diagnosis are particularly important to improve the quality of life and prolong life. The construction method is similar to the above and will not be described again.

Ribosomal display single-chain antibody library

Ribosomal display is a new technology for the screening and identification of functional proteins completely isolated, avoiding the drawbacks of traditional in vivo screening techniques, resulting in increased library capacity and enhanced molecular diversity. It binds the correctly folded protein and its mRNA to the ribosome, forming an mRNA-ribosomal-protein trimer that links the genotype and phenotype of the protein of interest. In recent years, ribosome display technology is built on a single-chain antibody library and applications have made great progress. The main processes of ribosome display technology include:

  • Construction of ScFv ribosome display template
  • ScFv single-chain antibody in vitro transcription and translation
  • Affinity screening and enrichment of ribosomal complexes
  • Evaluation of screening efficiency
  • In vitro evolution of ScFv molecules

The ribosome display technology is completely in vitro, and has the advantages of simple database construction, large library capacity, and simple screening method, no need to select pressure, and the like, and the introduction of mutation and recombination technology to improve the affinity of the target protein, so it is a large-scale construction. Because it can screen high-affinity protein molecules in a short period of time, its emergence opens up a new way to prepare small molecule antibodies. It is believed that with the continuous solution of shortcomings such as system stability in ribosome display technology, it will have great application value for the construction of antibody libraries and the screening of small molecule antibodies.

Anti-canine parvovirus single-chain antibody library

According to the larger bacterial display technology of bacterial particles, antibodies can be displayed on the surface of bacteria and the binding of antigen and antibody can be monitored by flow cytometry (FCM) in real time by fluorescent labeling, and the antibody-expressing strain with high affinity can be sorted. Using bacterial endometrial display technology, the foreign gene is expressed together with the NlpA signal peptide, and the target protein is displayed in the bacterial inner membrane. The NlpA signal peptide can transport the fusion gene of interest to the bacterial periplasmic space, and utilizes 6 specific amino acids to form a serotonin bond with the outer membrane of the bacterial endometrium during the transmembrane process, anchoring to the outside of the bacterial inner membrane to form a bacterial display antibody library. The outer membrane of the bacteria is treated with lysozyme, and the antibody library is screened by fluorescent labeling specific antigen binding to FCM. The method utilizes a 6 amino acid short peptide to anchor the foreign protein, has no effect on the conformation of the expressed antibody protein, and maintains genotype and phenotypic association. The aim is to screen recombinant antibodies with affinity anti-CPV gene, and lay a foundation for neutralizing active antibodies in subsequent studies. The neutralizing antibodies can bind to neutralizing epitopes on the virus, prevent the virus from binding to the corresponding receptors on the cell surface, and prevent diseases.

Evaluation

The ultimate goal of building a library is to screen out various specific antibody molecules. Whether the antibody of interest can be screened, in addition to the affinity of the antibody of interest and the target antigen, the quality of the library is also a determining factor. A library of antibodies with greater capacity and diversity will ensure the success of the screening. In general, the storage capacity is positively correlated with the affinity of the antibodies screened. High-affinity antibodies can only be screened in a high-capacity antibody library. At the same time, it must have good diversity. For natural antibody libraries, a wider range of antibody gene sources can better ensure the diversity of antibody libraries.

References

[1] Baca M, Presta LG, O 'Connor SJ, et a1. Antibody humanization using monovalent phage display [J].  J Biol Chem, 1997, 272:10678-10682.

[2] Lowenfels A B, Maisonneune P. Epidwminology and prevention of pancreatic cancer [J], Jpn J Clin Oncol, 2004, 349(5):238-242


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