Cloning and Quantification of Ferret Serum Amyloid A

Abstract

ABSTRACT. Serum amyloid A (SAA) is used as a biomarker for infections and inflammation in humans and veterinary medicine. We cloned ferret cDNA encoding SAA from the liver of a ferret via reverse transcription PCR (RT-PCR). The sequence of the cDNA clone revealed that ferret SAA has an open reading frame of 387 bp that encodes 129 amino acids. The deduced amino acid sequence of ferret SAA has 96. 1, 89.9, 86.0, 83.8, 83.0, 73.8 and 65.3% similarity to the mink, dog, cat, cattle, horse, human and mouse SAA genes, respectively. Compared to human SAA, the deduced ferret SAA amino acid sequence had an insertion of an 8-amino acid fragment between amino acids 88 and 95. Recombinant ferret SAA (rfrSAA) was expressed using an Escherichia coli(E. coli) strain, BL21 Star. Using Western blot analysis, anti-SAA mAb provided with the multispecies SAA ELISA kit reacted with purified rfrSAA. A significant dose-response relationship was observed between the rfrSAA protein and a commercial multispecies SAA ELISA kit. In contrast, rfrSAA was not recognized with the antibodies included in a commercial human SAA ELISA kit. These results suggest that the structure of ferret SAA is antigenically similar to other domestic animal SAAs, and the multispecies ELISA kit allows for the detection and quantification of ferret SAA in vivo. KEY WORDS: ELISA, ferret, serum amyloid A (SAA).