Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin

Vitellogenin (vtg) is a high molecular weight glycophospholipoprotein synthesized in the liver under stimulation of estrogen. Basically found in sexually mature female,vtg is taken up by developing oocytes during maturation. It functions as a nutrient storage for growing embryo. Vtg has the potentia...

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Main Author:	Muhammad, Nurul Ashikin
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Fishes - Breeding - Malaysia Fishery technology - Malaysia Freshwater fishes - Malaysia
Online Access:	http://psasir.upm.edu.my/id/eprint/49397/1/FP%202013%2046RR.pdf
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spelling	my-upm-ir.493972016-11-17T03:57:37Z Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin 2013-02 Muhammad, Nurul Ashikin Vitellogenin (vtg) is a high molecular weight glycophospholipoprotein synthesized in the liver under stimulation of estrogen. Basically found in sexually mature female,vtg is taken up by developing oocytes during maturation. It functions as a nutrient storage for growing embryo. Vtg has the potential as a maturation indicator for the successful production of fish fry. Tor tambroides is one of the most sought after fish in Malaysia, used as a game fish and economically important as cultured species. Main problem in the mass production of T. tambroides fry in hatchery is in the availability and selection of matured broodstock. Identification of matured and ready females morphologically can only be done by experience workers. Therefore,establishment of simple technique to identify matured females is necessary. Hence,in this current study, enzyme linked immunosorbent assay (ELISA) has been developed to measure blood plasma vtg as female maturation indicator. The development of this indicator will definitely contribute to the hatchery production of T. tambroides fry. Estrogen (17β-estradiol) was injected intra-peritoneally into five males (1.5 ±0.4kg).In order to confirm the synthesis of vitellogenin, raw plasma from E2-treated male, vitellogenic female and non-treated male were subjected to SDS-PAGE analysis.The presence of 149kDa protein band in E2-treated male plasma indicated the secretion of vitellogenin. Plasma samples were purified by size exclusion chromatography to separate protein particles according to molecular sizes. Fractions containing the bell-shaped major peaks were collected and subjected to native PAGE. Molecular weight of protein bulk was determined as 700kDa band. Further reduction of the protein bulk by SDS-PAGE resulted in the appearance of protein bands with similar positions in E2-treated sample and female whereas nontreated male showed no similarity. Specificity of antibody in Western blot revealed that in purified E2-treated male plasma, only three bands (133kDa, 117kDa, 56kDa) were recognized by anti-carp monoclonal antibody and thus identified as vtg. Bands with similar positions were also detected in mature female plasma. Male plasma did not show any cross reactivity against antibody. Enzyme-linked immunosorbent assay (ELISA) was developed for measurement of vtg concentration at plasma level. Purified vtg (253 ng/ml) was coated on 96-well microplate. Plasma samples were diluted with 1:500 antiserum to a final dilution of 1:1000. Linearization of binding displacement curves by logit transformation revealed that serial dilutions of mature female mahseer plasma slope was not statistically different from purified vtg of mahseer standard (F0.05=1.678 (12,28),p>0.05). ELISA assay developed for T. tambroides vitellogenin was confirmed through inter- and intra-assay validation. At different binding percentages (20, 50 and 80%), the coefficient of variation (CV%) of both precision assays (inter- and intra-assay) were less than 15% which means ELISA developed for the measurement of plasma vtg concentration in T. tambroides is sensitive and repeatable. Fishes - Breeding - Malaysia Fishery technology - Malaysia Freshwater fishes - Malaysia 2013-02 Thesis http://psasir.upm.edu.my/id/eprint/49397/ http://psasir.upm.edu.my/id/eprint/49397/1/FP%202013%2046RR.pdf application/pdf en public masters Universiti Putra Malaysia Fishes - Breeding - Malaysia Fishery technology - Malaysia Freshwater fishes - Malaysia
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Fishes - Breeding - Malaysia Fishery technology - Malaysia Freshwater fishes - Malaysia
spellingShingle	Fishes - Breeding - Malaysia Fishery technology - Malaysia Freshwater fishes - Malaysia Muhammad, Nurul Ashikin Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
description	Vitellogenin (vtg) is a high molecular weight glycophospholipoprotein synthesized in the liver under stimulation of estrogen. Basically found in sexually mature female,vtg is taken up by developing oocytes during maturation. It functions as a nutrient storage for growing embryo. Vtg has the potential as a maturation indicator for the successful production of fish fry. Tor tambroides is one of the most sought after fish in Malaysia, used as a game fish and economically important as cultured species. Main problem in the mass production of T. tambroides fry in hatchery is in the availability and selection of matured broodstock. Identification of matured and ready females morphologically can only be done by experience workers. Therefore,establishment of simple technique to identify matured females is necessary. Hence,in this current study, enzyme linked immunosorbent assay (ELISA) has been developed to measure blood plasma vtg as female maturation indicator. The development of this indicator will definitely contribute to the hatchery production of T. tambroides fry. Estrogen (17β-estradiol) was injected intra-peritoneally into five males (1.5 ±0.4kg).In order to confirm the synthesis of vitellogenin, raw plasma from E2-treated male, vitellogenic female and non-treated male were subjected to SDS-PAGE analysis.The presence of 149kDa protein band in E2-treated male plasma indicated the secretion of vitellogenin. Plasma samples were purified by size exclusion chromatography to separate protein particles according to molecular sizes. Fractions containing the bell-shaped major peaks were collected and subjected to native PAGE. Molecular weight of protein bulk was determined as 700kDa band. Further reduction of the protein bulk by SDS-PAGE resulted in the appearance of protein bands with similar positions in E2-treated sample and female whereas nontreated male showed no similarity. Specificity of antibody in Western blot revealed that in purified E2-treated male plasma, only three bands (133kDa, 117kDa, 56kDa) were recognized by anti-carp monoclonal antibody and thus identified as vtg. Bands with similar positions were also detected in mature female plasma. Male plasma did not show any cross reactivity against antibody. Enzyme-linked immunosorbent assay (ELISA) was developed for measurement of vtg concentration at plasma level. Purified vtg (253 ng/ml) was coated on 96-well microplate. Plasma samples were diluted with 1:500 antiserum to a final dilution of 1:1000. Linearization of binding displacement curves by logit transformation revealed that serial dilutions of mature female mahseer plasma slope was not statistically different from purified vtg of mahseer standard (F0.05=1.678 (12,28),p>0.05). ELISA assay developed for T. tambroides vitellogenin was confirmed through inter- and intra-assay validation. At different binding percentages (20, 50 and 80%), the coefficient of variation (CV%) of both precision assays (inter- and intra-assay) were less than 15% which means ELISA developed for the measurement of plasma vtg concentration in T. tambroides is sensitive and repeatable.
format	Thesis
qualification_level	Master's degree
author	Muhammad, Nurul Ashikin
author_facet	Muhammad, Nurul Ashikin
author_sort	Muhammad, Nurul Ashikin
title	Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
title_short	Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
title_full	Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
title_fullStr	Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
title_full_unstemmed	Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin
title_sort	purification and characterization of malaysian mahseer (tor tambroides bleekers) vitellogenin
granting_institution	Universiti Putra Malaysia
publishDate	2013
url	http://psasir.upm.edu.my/id/eprint/49397/1/FP%202013%2046RR.pdf
_version_	1747812014093238272

Purification and characterization of Malaysian Mahseer (Tor tambroides bleekers) vitellogenin

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