Immunobiochemical Characterization of Native Leptin from Goat ( Capra hircus ): Serodiagnostic Potentiality Revealed

摘要

The leptin, a cytokine–like adipose tissue–derived protein hormone, is one of the best physiological markers for body weight, food intake, energy expenditure and reproduction. Information regarding molecular characteristics of leptin protein in different animal species is scarce. With the aim of characterizing the leptin protein of Black Bengal goat at molecular level, the present study was designed to isolate the goat leptin protein from its adipocytes. In the present study, chemical treatment and ultra–sonication techniques were used for isolating leptin protein from goat adipose tissue. Purification of the protein was attempted using affinity column chromatography. The molecular, biophysical and serological characterization of leptin protein from Black Bengal goat were performed by 2D–gel electrophoresis, SDS–PAGE, MALDI–TOF mass spectroscopy and Western blotting. The 2D–gel electrophoresis analysis revealed that goat leptin possessed isoelectric point (P.I.) of 5.8 and molecular mass of 16 kDa. Western blot analysis revealed that the Black Bengal goat adipocytes possessed the seroreactive leptin protein of 16 kDa. MALDI–TOF mass spectroscopy and peptide analysis further confirmed the molecular mass of goat leptin to be of 15948.72 Da. The serodiagnostic potential of affinity purified protein was revealed while assessing its serological responses by dip–stick ELISA using anti–leptin antibodies that might be exploited while preparing of sero–diagnostic tool(s) for goat leptin detection from unknown (test) samples