Production of a Recombinant Single-Chain Variable-Fragment (scFv) Antibody against Sulfoglycolipid
| Autor: | Norihiro Kotani, Xiangchun Wang, Koichi Honke, Tae Watanabe, Yanglong Zhang, Tadashi Tai, Seung Ho Lee, Xinyao Cheng, Naoyuki Taniguchi, Ikuo Kawashima |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Male
Phage display medicine.drug_class Phagemid Molecular Sequence Data Immunoglobulin Variable Region Enzyme-Linked Immunosorbent Assay Immunoglobulin light chain Monoclonal antibody Biochemistry law.invention Mice law Testis medicine Animals Single-chain variable fragment Amino Acid Sequence Immunoglobulin Fragments Molecular Biology Mice Knockout Hybridomas Sulfoglycosphingolipids Base Sequence biology General Medicine Molecular biology Fusion protein Recombinant Proteins biology.protein Recombinant DNA Female Immunoglobulin Light Chains Glycolipids Sulfotransferases Antibody Immunoglobulin Heavy Chains |
| Zdroj: | Journal of Biochemistry. 137:415-421 |
| ISSN: | 0021-924X |
| Popis: | Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA. |
| Databáze: | OpenAIRE |
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