Single-chain antibody against human lipocalin-type prostaglandin D synthase: construction, expression, purification, and activity assay.
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.
Chen
De-Yu
DY
Research Center for Reproductive Medicine, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041, China.
Liu
Li-Min
LM
Liu
Sheng-Jie
SJ
Zhu
Mao-Ying
MY
Xu
Lan
L
Huang
Tian-Hua
TH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000276:immunology*; D054834:Lipocalins / Q000276:immunology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D015202:Protein Engineering
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase