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Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil.

ABSTRACT: Tonsillitis is the inflammation of the tonsils due to infection, many patients ultimately have to undergo tonsillectomy. In order to improve the accuracy of diagnosis and even create a new treatment for tonsillitis, we constructed a prokaryotic expression single-chain antibody fragment library against Streptococcus pneumoniae with immunoglobulin heavy chain variable region (VH), ? light chain (V?), and ? light chain (V?) genes by using human tonsil tissue. Plasmid DNA sequencing showed that single-chain antibodies were complete and constructed correctly. The binding activity of recombinant clones was detected by enzyme-linked immunosorbent assay (ELISA), results showed that the binding activity and specificity of anti-S. pneumoniae single-chain fragment variable (scfv) is proved to be successful. The single-chain antibody may be an attractive strategy for tonsillitis etiologic diagnosis and therapy.

SUBMITTER: Luo J

PROVIDER: S-EPMC6527077 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil.

Luo Jiaqi J Yang Lu L Long Zhen Z Xiao Zhiwen Z Sun Xiaomei X Zhuang Shimin S Xie Liangen L Wang Wei W Zhang Guanping G Qu Yue Y Liu Tianrun T

Bioengineered 20191201 1

Tonsillitis is the inflammation of the tonsils due to infection, many patients ultimately have to undergo tonsillectomy. In order to improve the accuracy of diagnosis and even create a new treatment for tonsillitis, we constructed a prokaryotic expression single-chain antibody fragment library against Streptococcus pneumoniae with immunoglobulin heavy chain variable region (VH), κ light chain (Vκ), and λ light chain (Vλ) genes by using human tonsil tissue. Plasmid DNA sequencing showed that sing ...[more]

PMID: 31088324

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Project description:BackgroundMonoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications.MethodsThe library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to β-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays.ResultsWe have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation.ConclusionThe promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Dataset Information

Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil.

Publications

Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil.

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