Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system

Sun, Dongbo; Shi, Hongyan; Chen, Jianfei; Da, Shi; Zhu, Qinghe; Zhang, Hong; Liu, Shengwang; Wang, Yunfeng; Qiu, Hua‐Ji; Feng, Li

doi:10.1016/j.jviromet.2012.03.021

Cited by 13 publications

(8 citation statements)

References 28 publications

(27 reference statements)

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“…Unlike other phage display systems, the T7Select system is capable of displaying non-membrane proteins. In addition, due to the small size of scFv PCR products, non-membrane proteins can be well presented in the T7Select system (18)(19)(20)(21). After four rounds of biopanning, specific anti-venom scFv presenting phages were enriched.…”

Section: Discussionmentioning

confidence: 99%

Functional recombinant single‑chain variable fragment antibody against Agkistrodon acutus venom

et al. 2019

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Agkistrodon acutus bites are conventionally treated with animal-derived antivenom, the use of which is limited due to allergic reactions and serum sickness. Thus in the present study, the genes of humanized antibodies produced in response to A. acutus venom were extracted from lymphocytes from patients bitten by A. acutus. A single-chain variable fragment (scFv) library against venom was constructed using a T7 phage display system. ScFv genes that exhibited high affinity to venom were selected by library biopanning. An expression system was constructed for antivenom scFv fused with 6xHis tag at its N-and C-terminus using pET-28a (+) vector. The scFv proteins could achieve functional and soluble expression in Escherichia coli via the auto-induction method. The purity and activity of the scFv genes and proteins were confirmed by SDS-PAGE, western blotting and ELISA. The results indicated that three soluble scFv proteins exhibited specific affinity to A. acutus venom and were harvested via the auto-induction method.

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Section: Discussionmentioning

confidence: 99%

Functional recombinant single‑chain variable fragment antibody against Agkistrodon acutus venom

et al. 2019

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“…The small molecules binding to pAPN, which have potential use for blocking TGEV infection in vitro, have become interesting targets for TGEV prevention and therapy. (9)(10)(11)(12)(13)(15)(16)(17) In the current study, five phage clones with a binding affinity to the rpAPN-C protein were obtained from the Ph.D.-12 phage display library by biopanning. The phage clone PM1 (HDAISWTHYHPW) showed a protective effect against TGEV infection in ST cells.…”

Section: Discussionmentioning

confidence: 99%

Screening and Antiviral Analysis of Phages That Display Peptides with an Affinity to Subunit C of Porcine Aminopeptidase

Guo

Zhu²,

Feng³

et al. 2013

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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The purified C subunit of the recombinant porcine aminopeptidase N (rpAPN-C) protein was used as an immobilized target to screen potential ligands against rpAPN-C from a 12-mer phage display random peptide library. After five rounds of biopanning, five phage clones showed specific binding affinities to rpAPN-C. In 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays, the phage clone PM1, which contained the HDAISWTHYHPW peptide sequence, had a protective effect against TGEV infection in swine testis cells. Therefore, the HDAISWTHYHPW peptide sequence has a potential use as a small molecular therapeutic agent against TGEV infection.

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“…In phage display systems, different bacteriophages have been used to display a range of proteins on surface. M13 filamentous bacteriophage [51,52] and T7 lytic phage are the most commonly used for displaying and production of antibody fragments [53,54]. A comparison between M13 bacteriophage and T7 lytic phage are discussed in the following section and summarized in Table 1.…”

Section: Types Of Bacteriophage Utilized In Phage Display Systemmentioning

confidence: 99%

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

Leow¹,

Cheng²,

Fischer³

et al. 2018

Antibody Engineering

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Monoclonal antibodies have become increasingly accepted as diagnostics and therapeutics for various human diseases due to their high affinity and specificity. However, several practical drawbacks are apparent for the reagents based on conventional IgG antibodies. With the emergence of antibody engineering, many problems were overcome when the recombinant antibody fragments such as Fabs, scFvs, diabodies and single domain antibodies (sdAbs), are developed. These fragments not only retain the specificity of the whole monoclonal antibodies, but are also easy to express and produce in prokaryotic expression systems. Rather unexpectedly, the natural sdAbs namely V HH s, V NAR s and variable lymphocyte receptors (VLRs) that comprise excellent biological activities were recently discovered in camelids, cartilaginous fish and lampreys, respectively. Due to their unique characteristics, including small size, high thermostability, stable folding in the nucleus and cytosol and long CDR3 regions which have access to cavities or clefts on the surface of proteins, these new binders are now investigated extensively as a substitute for conventional antibodies. This review describes the potential of sdAbs selected using in vitro display systems and their use in multiple applications.(polyclonal Abs) are heterogeneous antibody mixtures that are derived from multiple plasma cell lines. Because polyclonal antibodies comprise a mixture of different antibodies carrying numerous paratopes, they have excellent properties for recognizing antigens [2]. A monoclonal antibody (mAb) is a homogeneous antibody generated from a single B lymphocyte clone. Antibodies produced in mAb format have an extremely high specificity against a single epitope on antigens [3]. Recombinant antibodies (rAbs) are antibodies generated using molecular biology techniques. They are aimed to improve the sensitivity, selectivity, stability and immobilization properties in diagnostic applications, for example, in biosensors [4]. In making decision to use or generate polyclonal, monoclonal or recombinant antibodies, several factors should be considered, including commercial availability, possibility to raise animals, types of applications, time length of a project and costs [1]. Although a vast number of rAbs has been proposed [5][6][7][8], the natural sdAb fragments that were recently discovered from camelids (V HH s), sharks (V NAR s) and lampreys (VLRs) have shown to possess extraordinary features that are not found in conventional antibodies, such as a small dimension, an elevated stability and the capability of recognizing cavities and clefts on the surface of proteins that cannot be reached by conventional recombinant antibodies [9][10][11]. This chapter will discuss the availability of new binders derived from vertebrates and give an overview of their applications in a biomedical platform by recognizing specified targets from various diseases. Monoclonal antibodies and their limitationsThe first description of monoclonal antibody (mAbs) production was published ...

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Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system

Cited by 13 publications

References 28 publications

Functional recombinant single‑chain variable fragment antibody against Agkistrodon acutus venom

Functional recombinant single‑chain variable fragment antibody against Agkistrodon acutus venom

Screening and Antiviral Analysis of Phages That Display Peptides with an Affinity to Subunit C of Porcine Aminopeptidase

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

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