• Applied Biological Chemistry
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• v.49 no.3
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• pp.196-201
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• 2006

Vitellogenin, which is found in the serum of female and male fishes exposed to environmental endocrine disrupter or estrogen hormone, is used as a biomarker for environmental contamination with an endocrine disrupter. In order to produce antibody against vitellogenin, a synthetic peptide for partial vitellogenin was injected into rabbits. In addition, by using ion exchange chromatography on DE-52, vitellogenin was purified from the serum of carp induced with $17{\beta}$-estradiol. Polyclonal antibody against purified vitellogenin reacted well with vitellogenin in the serum of carp induced with $17{\beta}$-estradiol and the serum of female carp, whereas polyclonal antibody against the vitellogenin peptide did not react with proteins in those samples. This may indicate that vitellogenin proteins, covalently modified largely, could not be detected by Western blotting with the polyclonal antibody against the synthetic vitellogenin peptide.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.6
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• pp.753-760
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• 1995

This study was conducted to determine the serum vitellogenin (VTG) concentration changes during the ovulation period in elkhorn sculpin, Alcichthys alcicornis. The results of sepacryl S-300 showed that the molecular weight of VTG could be 380,000. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis may indicate that the purified VTG consists of three subunits with molecular weights of 180,000, 118,000 and 85,000, respectively. Yolk protein purified from the egg extracts was eluted on an equilibrated sephacryl S-300 column, and its molecular weight was estimated 250,000. The precipitation lines of the female serum against the antiserum of the egg extracts were fused completely by immunoelectrophoresis and immunodiffusion analysis. VTG was detected in the serum, and hepatocytes from males injected with $17\beta-estradiol\;(E_2).$ Furthermore, VTG was immunochemically similar to yolk proteins. The concentration of VTG was high before ovulation $(9.80\pm0.81-11.02\pm0.09 mg/ml),$ and then decreased rapidly after ovulation $(less\;than\;6.19\pm0.59 mg/ml).$ This study suggested that VTG was synthesized in the liver by the action of $E_2$ and released to blood, and then incorporated into oocytes.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2002.05a
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• pp.171-171
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• 2002

• Journal of Sericultural and Entomological Science
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• v.31 no.2
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• pp.72-81
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• 1989

Antheraea yamamai vitellin was purified from matured eggs by polyacryamide gel electrophoresis, also stage dependent appearance, immunological comparison and relative content of the protein were investigated. 1. Vitellogenin, the precursor of vitellin, was first detected in the larval hemolymph at the late spinning stage by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The electrophoretic mobility of the vitellin was identical with that of Bombyx mori and of Bombyx mandarina. However, the specific antiserum against A. uamamai vitellin did not react with either that of Bombyx mori or Bombyx mandarina in immumo-diffusion test. 3. Relative content of A. yamamai vitellin to the total soluble egg protein was 46.0 percent and did not change till eight days after oviposition. But the content started to decline from ten days after oviposition and was negligible in the five or serventeen month old eggs.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.99-108
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• 2004

Vitellogenin (VTG) was purified from serum of $estradiol-l7{\beta}-treated rockfish$(Sebastes schlegeli) by precipitation with $EDTA-Mg^{2+}$ and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188 kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of $estradiol-l7{\beta}-treated rockfish$ treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2 ng/mL and 1,000 ng/mL and the value of the intra and inter assay variations were within $9.7{\%}$ and $11.2{\%}$, respectively. Recovery rate was $96.8{\%}$. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.

• Journal of Sericultural and Entomological Science
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• v.30 no.2
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• pp.96-104
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• 1988

Vitellin, the major yolk protein of the silkworm, Bombyx mori was pruified, and its immunological properties and the quantitative changes during embryogenesis were studied. The ovary transplantation into male hosts was also carried out to find its effect on the yolk protein synthesis. The pupal vitellogenin and the egg vitellin of Bombyx mori were purified by DEAE-cellulose column chromatography. These two female specific proteins showed the same mobility in the polyacrylamide gel electrophoresis and the same reaction in the double immunodiffusion test. The immunological identity was also observed between the vitellins of Bombyx mori and Bombyx mandarina. The rudimentary ovaries transplanted into the male hosts of silkworms produced eggs without vitellin, indicating that the yolk precursors synthesized in other female organ beyond the ovary were necessary to produce vitellins. The major yolk protein, vitellin was disintegrated and utilized mostly during late stage of embryogenesis. It was different characteristics from the egg specific protein, which was utilized continuously from the early embryonic stage.

• Fisheries and Aquatic Sciences
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• v.8 no.4
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• pp.213-219
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• 2005

Vitellogenin (VTG) was purified from the blood plasma of estradiol-17$\beta$ ($E_2$)-treated male marbled sole (Limanda yokohamae) using gel filtration and anion exchange chromatography. The purity of the marbled sole VTG (msVTG) was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing. The purified msVTG was used to produce monoclonal and polyclonal antibodies in mice and rabbits, respectively, and the specificity of the polyclonal antisera for msVTG was confirmed by Western blot analysis. The antibodies cross­reacted with a protein of molecular mass approximately 160 kDa in the plasma samples of mature female marbled sole. No cross-reactivity was observed with the plasma of male fish. A direct non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed using the monoclonal anti-msVTG and polyclonal anti-msVTG antibodies, with purified msVTG as the standard protein. The values of the intra- and inter-assay variations were within the ranges of $8.l-9.8\%$ and $8.5-12.2\%$, respectively. The sensitivity was about 0.3 ng/mL. Serial dilutions of plasma from mature female sole reacted with the msVTG-antibodies in the sandwich ELISA, whereas the plasma from male fish did not. The results indicate that the maturation status of female marbled sole can be identified using a sandwich ELISA for msVTG.

• Journal of Aquaculture
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• v.21 no.4
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• pp.285-293
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• 2008

Vitellogenin (Vg) is the precursor of vitellin (Vn), the major yolk protein of teleost fishes. In this study, Vg and Vn proteins of the Korean bullhead Pseudobagrus fulvidraco were isolated using gel-filtration chromatography (Sephadex-G 200 column) and anion-exchange chromatography (Mono Q HR 5/5 column), respectively. Purified Vn with an estimated molecular mass of 360 kDa by gel filtration chromatography was obtained from ovarian egg, and it was composited to one major subunit with an estimated molecular mass of 107 kDa by SDS-PAGE. In the result of western blotting, one major band was detected using antiserum against Vn (anti-Vn). These results suggested that Vn was composed of three subunits having the same molecular weight in Pseudobagrus fulvidraco. Vg was induced by estradiol-$17{\beta}$ ($E_2$) and purified from $E_2$ treated male serum. The molecular weight of whole Vg was estimated to be 450 kDa by gel filtration chromatography, and it is composed of three subunits with estimated molecular masses of 110 kDa, 125 kDa and 147 kDa as determined by SDS-PAGE. In the Ouchterlony's immunodiffusion test using anti-Vn and antiserum against female and male serum, purified Vg was detected in matured female and Ez treated male serum but not in untreated male. These results can be used in detecting estrogenic contamination of the aquatic environment.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.5
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• pp.432-442
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• 1992

To identify the histological site of synthesis of yolk protein precursor, vitellogenin, by immunocytochemical method in the freshwater crab Eriocheir japonicus, we purified the yolk protein, vitellin, from crude egg extracts, and prepared the anti-rabbit serum against vitellin. Then, the site of vitellogenin synthesis was demonstrated by immunotytochemical method with PAP(peroxidase-antiperoxidase) reaction using the rabbit antiserum aganist vitellin. Female specific serum protein was identified in female serum by immunoelectrophoresis and Ouchterlony's immunodiffusion test for mature male and female sera. Based on the immunoelectrophoresis and Ouchterlony's diffusion test for mature male and female sera and crude egg extracts using antiserum against vitellogenic female serum absorbed with male serum, the female specific serum protein was identified as vitellogenin, detected in female serum only. The major yolk protein, vitellin, was purified from the crude egg extracts by DEAE-cellulose ion exchange chromatography, followed by sepharose CL-4B gel filteration chromatography. The molecular weight of vitellin was estimated to be about 245,000 dalton by sepharose CL-4B gel filteration chromatography. from the results of immunological analysis for vitellin, it was found that the vitellin antiserum contained the antibody against vitellogenin. In the results of immunocytochemical reaction by PAP method with the rabbit antiserum against vitellin, the vitellogenic oocytes and the hepatopancreas of mature female showed positive PAP reaction, but not in follicle cells and previtellogenic oocytes nf ovary, muscle of female and mature male hepatopancreas. Therefore, it showed that the hepatopancreas of mature female is the site of vitellogenic synthesis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.3
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• pp.473-479
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• 1997

The yolk protein of spotted flounder, Verasper variegatus was purified by precipitation wit cold distilled water, followed by Sepharose CL-6B column chromatography. The purified protein was identified as vitellin by Ouchterlony's immunodiffusion test and immunoelectrophoresis. The purified vitellin from ovarian crude extracts has same antigenic determinants with the female specific serum protein, vitellogenin. The molecular weight of purified vitellin was estimated about 550 kD by gel filfration. The vitellin was composed of three major subunits with molecular weight of about 108, 85 and 31 kD, and two minor subunits. The vitellin was identified by western blot analysis using anti-vitellin antibody.