Recombinant feline oviductin--a powerful tool for functional IVF studies in the domestic cat.

摘要: Contents Oviductins are highly conserved proteins that seem to play an important role during fertilization and embryo development. A universal characteristic of oviductin is its association with the zona pellucida ovulated oocytes, several functional studies indicate influence this glycoprotein on reproductive events in mammals. The objective study was produce recombinant feline prokaryotic as well eukaryotic cells a prerequisite for analysing IVF domestic cat. Oviductin sequence cloned into pET21b vector transformed E. coli His-tagged, non-glycosylated protein. isolated from lysate using anion exchange chromatography followed by immobilized metal ion affinity (IMAC). Western blot analysis purified fractions resulted one clear band corresponding expected size ~67 kDa. coomassie-stained gel analysed mass spectrometry. identified over 50 peptides covering 72% whole protein sequence. To obtain glycosylated form oviductin, were stable transfected pSecTag/HygroA containing gene His-tagged harvested serum- protein-free cell culture medium. Mass spectrometry analyses bands obtained after separation medium SDS-PAGE detected ~70, ~85 ~170 kDa. With produced we now able use further