A method for high-throughput screening of single-domain antibodies using ispla and its application

Abstract

The invention discloses a method for high-throughput screening of single-domain antibodies using isPLA. The steps are as follows: constructing an sdAb library containing 21 random amino acid sequences in the CDR3 region, and fusing a 3×Flag tag to the C-terminus of the sequence; co-transfection Cells, fixed cells for isPLA; followed by sorting, amplification, and recombination, and after multiple rounds of screening and sequencing, constructs that sequentially contain glutathione S-transferase GST, tobacco mosaic virus TEV protease cleavage site, and recognize SQSTM1. The sdAb sequence, the translocation domain of Pseudomonas exotoxin ETA and the 3×Flag-tagged recombinant protein were expressed in E. coli, followed by purification and enzyme digestion. The method can screen sdAbs that specifically recognize different antigenic determinants from the constructed high-capacity sdAb library, and has low cost and high efficiency. No preparation of animal samples or hybridomas is 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 that there are two types of immunoglobulins in camelid serum: 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 by cloning the variable region of the heavy chain of the antibody, a single-domain antibody (single-domain antibody, sdAb, also known as nanobody, nanobody) can be obtained 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, prepare and carry out genetic engineering. , 2018, 14(1):1-19.). For example, it can act as a molecular chaperone during protein crystalliz...

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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