Single-Chain Antibody against Human Lipocalin-Type Prostaglandin D Synthase: Construction, Expression, Purification, and Activity Assay

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De-Yu Chen^1,2, Li-Min Liu¹, Sheng-Jie Liu², Mao-Ying Zhu², Lan Xu¹, and Tian-Hua Huang^1*

¹Research Center for Reproductive Medicine, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, Guangdong, China; E-mail: thhuang@stu.edu.cn

²Department of Biology, Fuyang Normal College, Fuyang 236032, China

^* To whom correspondence should be addressed.

Received September 20, 2007; Revision received January 5, 2008

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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.

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KEY WORDS: prostaglandin D synthase (L-PGDS), single-chain antibody (ScFv), expression

DOI: 10.1134/S0006297908060114

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