Molecular cloning, expression in Escherichia coli, and development of monoclonal antibodies to feline cystatin C

Abstract

Cystatin C is a member of the cystatin superfamily of low-molecular-weight proteins that inhibit the activity of cysteine protease. The full-length cDNA that encodes feline cystatin C (FeCysC) has been cloned from feline white blood cells by the oligo-capping method. The cDNA consists of 796 nucleotides and includes an open reading frame encoding a polypeptide of 146 amino acids. Next, we developed several mouse anti-FeCysC monoclonal antibodies (mAbs). The recombinant FeCysC (rFeCysC) was expressed in Escherichia coli (E. coli) BL21 and used as an antigen to immunise mice. The reactivity of the mAbs to native FeCysC was examined by western blot analysis against the urinary protein from cats with chronic kidney disease (CKD). The three anti-rFeCysC mAbs (3-9G, 7-7C, and 9-12F) were able to recognise native FeCysC.