1. Single-chain antibody against human lipocalin-type prostaglandin D synthase: construction, expression, purification, and activity assay.
- Author
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Chen DY, Liu LM, Liu SJ, Zhu MY, Xu L, and Huang TH
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Tumor, Cloning, Molecular, Escherichia coli, Gene Expression, Humans, Immunoglobulin Fragments genetics, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains metabolism, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains metabolism, Mice, Models, Biological, Molecular Sequence Data, Immunoglobulin Fragments biosynthesis, Immunoglobulin Fragments isolation & purification, Immunoglobulin Fragments pharmacology, Intramolecular Oxidoreductases immunology, Lipocalins immunology, Protein Engineering
- Abstract
An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.
- Published
- 2008
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