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Generation of Selenium-containing Human Single-chain Fv Antibody with GPX Activity

Jing Wei

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Abstract

Human single chain fragments variable(scFvs) against GSH were screened from a phage display human scFv antibody library. After three rounds of panning, five clones(2, 11, 16, 24, 32 ) binding to GSH were selected by ELISA. Analysis of PCR products using gel electrophoresis and sequencing showed that three clones(11, 16, 24) contained intact scFv-encoding gene. The scFv-encoding gene from clone 11 was subcloned into the expression vector pPELB and expressed as soluble form(scFv-11) in Escherichia coli Rosetta. The scFv-11 was purified by Ni2+-immobilized metal affinity chromatography. The specificity to GSH of scFv-11 was verified by immuno-dot blot. Selenium-containing human scFv(Se-scFv-11) with GPX activity of 351 U/mol was obtained by the chemical modification of the reactive serine residues into Secs.

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What this paper is about

Human single chain fragments variable(scFvs) against GSH were screened from a phage display human scFv antibody library. After three rounds of panning, five clones(2, 11, 16, 24, 32 ) binding to GSH were selected by ELISA. Analysis of PCR products using gel electrophoresis and sequencing showed that three clones(11, 16, 24) contained intact scFv-encoding gene. The scFv-encoding gene from clone 11 was subcloned into the expression vector pPELB and expressed as soluble form(scFv-11) in Escherichia coli Rosetta. The scFv-11 was purified by Ni2+-immobilized metal affinity chromatography. The specificity to GSH of scFv-11 was verified by immuno-dot blot. Selenium-containing human scFv(Se-scFv-11) with GPX activity of 351 U/mol was obtained by the chemical modification of the reactive serine residues into Secs.

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Available abstract

Human single chain fragments variable(scFvs) against GSH were screened from a phage display human scFv antibody library. After three rounds of panning, five clones(2, 11, 16, 24, 32 ) binding to GSH were selected by ELISA. Analysis of PCR products using gel electrophoresis and sequencing showed that three clones(11, 16, 24) contained intact scFv-encoding gene. The scFv-encoding gene from clone 11 was subcloned into the expression vector pPELB and expressed as soluble form(scFv-11) in Escherichia coli Rosetta. The scFv-11 was purified by Ni2+-immobilized metal affinity chromatography. The specificity to GSH of scFv-11 was verified by immuno-dot blot. Selenium-containing human scFv(Se-scFv-11) with GPX activity of 351 U/mol was obtained by the chemical modification of the reactive serine residues into Secs.

Key concepts: Panning (audio), Molecular biology, Phage display, Single-chain variable fragment, Chemistry, Antibody, Affinity chromatography, clone (Java method)

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