Isolation, purification and identification of matrilysin protein from canine mammary tumours and its quantification by indirect ELISA Reddy A.P. Vishwanatha3, Kumar B.V. Sunil3, Ranganath G.J.1,3, Kumar K. Aswani3, Maiti S.K.2,3, Kataria Meena*,3 3Division of Biochemistry, Indian Veterinary Research institute, Izatnagar, Bareilly-243122 (U.P) 1division of Veterinary Pathology, Indian Veterinary Research institute, Izatnagar, Bareilly-243122 (U.P) 2Division of Surgery and Radiology, Indian Veterinary Research institute, Izatnagar, Bareilly-243122 (U.P). *Corresponding author; e-mail: mkataria97@rediffmail.com
Abstract The extracellular matrix (ECM) consisting of proteoglycans, glycoproteins and glycosaminoglycans plays a fundamental role in the structural frame work to support cells and maintains cellular functions by mediating cell-cell or cell-ECM interactions. Matrix metalloproteinases (MMPs) play an important role in ECM degradation thereby favouring tumour progression. The purification of Matrilysin (MMP7) from canine mammary tumour was done by ammonium sulphate precipitation (40–80% saturation) followed by gel filtration using a Cellulofine GCL2000 column (2.6X98cm) (Chisso Co., Ltd, Japan). The peak fraction of gel filtration showed single band at 120 kDa on SDS-PAGE and cross reacted with human MMP7 polyclonal antibodies raised in goat on western blot analysis. On reduction with dithiothreitol treatment of peak fraction (120kDa), 28 kDa protein band was observed on SDS-PAGE. 20 kDa protein showed no cross reactivity with human MMP7 polyclonal antibodies. Results indicate that MMP7 of canine mammary tumour is tetramer of 28 kDa subunit. In dot blot-ELISA the purified 120 kDa fraction and sera of mammary tumour cases showed positive result as compared to normal and post operative serum. MMP-7 (120 kDa) was 9 times higher in mammary tumour infected serum as compared to normal serum and 4.7 times higher in mammary tumour tissue as compared to normal tissue by Indirect ELISA. Top Keywords Canine, ELISA, Matrilysin, Mammary tumour. Top |