Method for high-throughput screening of single-domain antibody by utilizing isPLA and application of method

Abstract

The invention discloses a method for high-throughput screening of a single-domain antibody by using isPLA, which comprises the following steps: constructing an sdAb library of a CDR3 region containing 21 random amino acid sequences, and fusing a 3*Flag tag at the C end of the sequence; co-transfecting the cells, and fixing the cells for isPLA; and then performing sorting, amplification and recombination, after multiple rounds of screening and sequencing, constructing the recombinant protein sequentially containing glutathione S-transferase GST, a tobacco mosaic virus TEV protease cleavage site, an sdAb sequence for identifying SQSTM1, a translocation structural domain for identifying pseudomonas exotoxin ETA and a 3*Flag tag, performing expression in escherichia coli, and then performing purification and enzyme digestion to obtain the recombinant protein. According to the method, the sdAbs capable of specifically recognizing different antigenic determinants can be screened from the constructed high-capacity sdAb library, the cost is low, and the efficiency is high. The preparation of animal samples or hybridomas is not involved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for high-throughput screening of single-domain antibodies using isPLA and its application. Background technique [0002] In 1993, Hamers-Casterman et al. found two types of immunoglobulins in the serum of camelids: one is a traditional antibody composed of two heavy chains and two light chains; the other is only two heavy chains chain (Nature. 1993; 363:446-448.). Only the heavy chain variable region of the antibody is cloned to obtain a single-domain antibody (single-domain antibody, sdAb, also known as nanobody, nanobody), with a molecular weight of about 15kD. sdAb has a stable structure, high affinity and antigen-binding ability, small relative molecular mass, easy to penetrate tissue barriers, easy to produce and carry out genetic engineering, and has received extensive research and attention due to its unique advantages (Journal of Biomedical Nanotechnology , 2018, 1...

AI Technical Summary

Problems solved by technology

The whole process takes a long time, more experimental materials are used, and the cost is higher, making the success rate of obtaining high-affinity sdAb extremely low

In addition, based on the amino acid sequence of the known antibody epitope, there are also some reports on engineered sdAb research, but these studies are a further supplement to the function of existing antibodies; and the attempt to find a new sdAb with higher specificity from scratch is still facing challenges. Big challenge, lack of high-affinity, high-specificity screening methods at the single-cell level, to be developed

[0005] Through the search, no patent publications related to the patent application of the present invention have been found

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