Immunobiochemical Characteristics of Purified Native Leptin Protein from Indian Major Carp, Rohu (Labeo rohita Ham.)


Information regarding molecular characteristics of leptin protein in different animal species in-cluding fish is scarce. With the aim of characterizing the native leptin protein of Indian major carprohu (Labeo rohita), at molecular level, the present study was designed to isolate rohu leptin from its hepatocytes (the prime source of leptin in fish) and immunobiochemical characterization of the same, subsequently. In the present study, chemical treatment and ultra-sonication technique was used for isolating leptin from rohu liver tissue. Purification of the protein was attempted using affinity column chromatography. The molecular, biophysical and serological characterization of rohu leptin was carried out by 2D-gel electrophoreis, SDS-PAGE, MALDI-TOF Mass spectroscopy and Western blot. The SDS-PAGE and 2D gel analysis revealed that rohu native leptin possesses molecular mass of 16 kDa. Western blot analysis showed that the fish hepatocytes possessed the sero-reactive leptin protein of 16 kDa. MALDI-TOF mass spectroscopy and peptide analysis showed the molecular mass of rohu leptin as 16283.38 Da. The serodiagnostic potential of native leptin of rohu was revealed for the first time while assessing its serological responses by ELISA using anti-leptin antibodies.

Share and Cite:

Alam, S. , Joardar, S. , Panigrahi, A. , Abraham, T. , Mukherjee, S. and Mukherjee, A. (2014) Immunobiochemical Characteristics of Purified Native Leptin Protein from Indian Major Carp, Rohu (Labeo rohita Ham.). Open Journal of Immunology, 4, 139-147. doi: 10.4236/oji.2014.44016.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Ahima, R.S. and Filer, J.S. (2000) Leptin and the Neuroendocrinology of Fasting. Annual Reviews of Physiology, 62, 413-437.
[2] Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopoid, L. and Friedman, J.M. (1994) Positional Cloning of the Mouse Obese Gene and Its Human Homologue. Nature, 372, 425-432.
[3] McGregor, G.P., Desaga, J.F., Ehlenz, K., Fischer, A., Heese, F., Hegele, A., Lammer, C., Peiser, C. and Lang, R.E. (1996) Radio Immunological Measurement of Leptin in Plasma of Obese and Diabetic Human Subjects. Endocrinology, 137, 1501-1504.
[4] Cohen, S.L., Halass, J.L., Friedman, J.M., Chait, B.T., Bennett, L., Chang, D., Hecht, R. and Collins, F. (1996) Human leptin Characterization. Nature, 382, 15-20.
[5] Geary, T.W., McFadin, E.L., MacNeil, M.D., Grings, E.E., Short, R.E., Funston, R.N. and Keisler, D.H. (2003) Leptin as a Predictor of Carcass Composition in Brief Cattle. Journal of Animal Sciences, 81, 1-8.
[6] Peinado, J.R., Jimenez-Gomez, Y., Pulido, M., Ortega-Bellido, M., Diaz-Lopez, C., Padillo, J.F., Lopez-Miranda, J., Vazquez-Martínez, R. and Malagon, M. (2010) The Stromal-Vascular Fraction of Adipose Tissue Contributes to Major Differences between Subcutaneous and Visceral Fat Depots. Proteomics, 10, 3356-3366.
[7] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein Measurement with Folin-Phenol Reagent. Journal of Biological Chemistry, 193, 265-275.
[8] Laemmli, U.K. (1970) Cleavage of Structural Proteins during Assembly of the Head of Bacteriphage T4. Nature, 227, 680-685.
[9] Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996) Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Annals of Chemistry, 68, 850-858.
[10] Mishra, S.S. and Sekhar, M.S. (1997) ELISA and Dot Immunoassay for Detection of Vibrio spp. in Tiger Shrimp Penaeus monodon. Indian Journal of Fisheries, 44, 369-376.
[11] Ouchterlony, O. (1953) Antigen-Antibody Reactions in Gels. IV. Types of Reactions in Coordinated Systems of Diffusion. Acta Pathologica et Microbiologica Scandinavica, 32, 231-240.
[12] Towbin, H., Staehelin, Τ. and Gorden, J. (1979) Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. Proceedings of National Academy of Science of USA, 76, 4350-4354.
[13] Mandal, S.M., Dey, S., Mandal, M., Neto, S.M. and Franco, O.L. (2009) Identification and Structural Insights of Three Novel Antimicrobial Peptides Isolated from Green Coconut Water. Peptides, 30, 633-637.
[14] Jiahao, M., Xueqin, L., Zhenyin, W., Zhenguo, Y. and Wendong, W. (1997) Rapid Detection of Motile Aeromonas Species in Fish by Dot ELISA. Journal of Dalian Fisheries College, 12, 72-78.
[15] Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L. and Friedma, J.M. (1995) Weight Reducing Effects of the Plasma Protein Encoded by the Obese Gene. Science, 269, 543-546.
[16] Cinti, S., Frederich, R.C., Zingaretti, M.C., De Matteis, R., Flier, J.S. and Lowell, B.B. (1997) Immunohistochemical Localization of Leptin Uncoupling Protein in White and Brown Tissue. Endocrinology, 138, 797-804.
[17] DiMarchi, R.D., Hermeling, R.N. and Hoffmann, J.A. (1998) Anti-Obesity Proteins. US Patent 5,7,19,266.
[18] Takahashi, N., Patel, H.R., Qi, Y., Dushay, J. and Ahima, R.S. (2002) Divergent Effects of Leptin in Mice Susceptible or Resistant to Obesity. Hormone Metabolism Research, 34, 691–697.
[19] Taniguchi, Y., Itoh, T., Yamada, T. and Sasaki, Y. (2002) Genomic Structure and Promoter Analysis of the Bovine Leptin Gene. IUBMB Life, 53, 131-135.
[20] Alam, S.S., Joardar, S.N. and Panigrahi, A. (2014) Immunobiochemical Characterization of Native Leptin from Goat (Capra hircus): Serodiagnostic Potentiality Revealed. Advances in Veterinary and Animal Sciences, 2, 86-90.
[21] Kochan, Z., Karbowska, J. and Meissner, W. (2006) Leptin Is Synthesized in the Liver and Adipose Tissue of the Dunlin (Calidris alpina). General and Comparative Endocrinology, 148, 336-339.
[22] Xu.,Q., Xinming, Z., Ning, H., Jianxin, S., Rong, H., Zhihe, Z., Hongwu, B. and Muyuan, Z. (2010) Characterization and Expression of Ailuropoda melanoleuca Leptin (ob Gene). Zoological Science, 27, 41-46.
[23] Kauter, K., Bal, M., Kearney, P., Tellam, R. and McFarlane, J.R. (2000) Adrenaline, Insulin and Glucagon Do Not Have Acute Effects on Plasma Leptin Levels in Sheep: Development and Characterisation of an Ovine Leptin ELISA. Journal of Endrocrinology, 166, 127-135.
[24] Shibata, H., Akahane, R., Honjoh, T., Asano, M., Mominoki, K., Fujii, K., Suzuki, M., Ohtashi, N., Ishioka, K., Ahmed, M., Soliman, M., Kimura, K. and Saito, M. (2005) Seasonal Changes in Serum Leptin of the Feral Raccoon (Procyon lotor) Determined by Canine-Leptin-Specific ELISA. Journal of Experimental Zoology, 303A, 527-533.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.