Method for synthesizing controllable humanized antibody library based on combinatorial synthesis technique and application

Abstract

The invention relates to a method for synthesizing a controllable humanized antibody library based on a combinatorial synthesis technique and application. The method comprises the following steps: synthesizing a section of sequence at a 3'-end by using a DNA solid phosphoramidite triester method till an amino acid change site is generated; collecting all intermediate products; mixing according to the requirements of the designed sequence, and grouping according to a ratio till controllability of change numbers is achieved; further synthesizing a corresponding codon sub basic group sequence till a next amino acid change site; collecting intermediate products, and remixing uniformly till another amino acid change site is generated; according to the requirements of the designed sequence, completing synthesis of a DNA sequence and a 5'-end overall length sequence to be synthesized; repeating the steps 1-3 to achieve possibility of any change of the generated amino acid change site and the number of any change. The method provided by the invention is high in controllability, high in flexibility and free of mutation, and the capacity of the synthesized humanized antibody library is large.

Description

technical field [0001] The invention belongs to the field of genetic engineering, especially combinatorial synthesis technology, and specifically relates to a method and application for synthesizing a controllable humanized antibody library based on combinatorial synthesis technology. Background technique [0002] Antibodies are produced by B lymphocytes after the body is stimulated by antigens. They can specifically bind or recognize invading pathogenic microorganisms. They are the most important immune molecules that exert immune functions in the humoral immune response and are mainly distributed in serum. Antigens often have a variety of different antigenic determinants, thereby stimulating the body to produce polyclonal antibodies; this kind of antibody is not uniform, which will affect the specificity and sensitivity of detecting antigens, and its clinical application is greatly limited. [0003] In the mid-1970s, B-lymphocyte hybridoma technology came out, and the mono...

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Problems solved by technology

[0007] Traditional monoclonal antibody production uses hybridoma technology to obtain stable monoclonal antibody strains through spleen cell fusion, which takes at least several months, and the screening range of monoclonal antibodies is relatively narrow, which seriously affects the preparation of high-affinity human monoclonal antibodies

Traditional monoclonal antibodies are all mouse-derived monoclonal antibodies. Although there have been many successful reports on the humanization of mouse antibodies, and there are also antibodies that have been approved for marketing, there are still the following problems: it is difficult to avoid human anti-mouse antibody reactions, and in the mouse antibody Decreased affinity and reduced stability during humanization

However, this method will introduce irrelevant mutations or stop codons, resulting in a significant decrease in the quality of the peptide library when a longer sequence needs to be synthesized, so it cannot be applied to situations where there are many mutation sites, or there are many types of amino acids involved in degenerate sites. Cannot meet the requirement of complete randomization

Some improved schemes, such as (NNK)n, (NNB)n and (VNN)n schemes, where N=A, T, C or G; K=T or G; B=G, C or T; V=A , G or C, to a certain extent, increased the diversity and reduced the participation of stop codons, but because the frequency of various codons encoding amino acids in each improved scheme was quite different, various subgroups in chemical synthesis Phosphate amide monomers will have certain differences in competitive activity during synthesis, making it difficult to control the amino acid composition bias of the synthesized sequence. This amino acid composition bias becomes more obvious as the length of the target sequence increases

Using polynucleotides (dinucleotides or trinucleotides) instead of single nucleotide monomers for synthesis, especially the synthesis of trinucleotides has outstanding advantages. It is synthesized in units of triplet codons, forming 20 kinds Synthetic units, using the random combination of these 20 triplet synthetic units to synthesize, can arbitrarily regulate the type and quantity of amino acids involved in specific sites, thereby avoiding sequence randomization and the addition of irrelevant amino acids and stop codons, but this The triplet raw material used in the method is very expensive, and the coupling efficiency of this raw material in chemical synthesis is very low, so it is difficult for most laboratories to realize in practice

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