[논문]Purification, Partial Characterization, and Immunoassay of Vitellogenin from Marbled Sole (Limanda yokohamae)

Kim Dae-Jung; Jung Jee-Hyun; An Cheul-Min; Jee Young-Ju; Min Kwang-Sik; Kim Yoon; Han Chang-Hee

doi:10.5657/fas.2005.8.4.213

Purification, Partial Characterization, and Immunoassay of Vitellogenin from Marbled Sole (Limanda yokohamae) 원문보기

Journal of fisheries science and technology, v.8 no.4, 2005년, pp.213 - 219

Kim Dae-Jung (Aquaculture Research Team, NFRDI) , Jung Jee-Hyun (South Sea Institute, KORDI) , An Cheul-Min (Aquaculture Research Team, NFRDI) , Jee Young-Ju (Aquaculture Research Team, NFRDI) , Min Kwang-Sik (Aquaculture Research Team, NFRDI) , Kim Yoon (Aquaculture Research Team, NFRDI) , Han Chang-Hee (Department of Molecular Biology, Dong-Eui University)

Abstract ▼ AI-Helper

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.

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문제 정의

The goal of this study was to develop a sensitive, quantitative ELISA for the detection of VTG in marbled sole. The first step in the development of the sandwich ELISA was to purify and characterize msVTG.

제안 방법

In this study, to further clarify the role of VTG in the sexual maturation of marbled sole (Limanda yokohamae), we purified and characterized the VTG of marbled sole and developed a sandwich ELISA for the quantification of plasma VTG levels using monoclonal and polyclonal antibodies against marbled sole VTG.
BALB/c mice were injected with purified msVTG (50 fig/mouse) that was emulsified in Freund's complete adjuvant (1:1). The mice were injected with 30 jig of msVTG in Freund's incomplete adjuvant (1:1) two times at two-week intervals. Three days before fusion of the spleen cell with the myeloma cell line (sp2/0-Agl4), the mice were boosted with 50 jig msVTG.

대상 데이터

M이e marbled sole (400-600 g body-weight) were acclimated for two weeks in the laboratory. VTG synthesis was induced by three intraperitoneal injections at 10-day intervals of 2 ㎎/㎏ body-weight estradiol-17P (E2; Sigma, UK), which was dissolved in ethanol-peanut oil.

이론/모형

Fractions that contained purified msVTG were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was performed according to the methods of Laemmli (1970). After electrophoresis, gel was fixed and stained with 0.

성능/효과

In conclusion, the 160-kDa protein band observed in the Ea-treated male fish is identified as the VTG of marbled sole based on: (1) the protein is observed exclusively in mature females; (2) the protein appears or is increased in level by E2 treatment of the male fish; and (3) the protein is similar in molecular weight and in N-terminal amino acid sequence to the VTGs of other species, such as the rainbow trout, Oncorhynchus mykiss (175-kDa VTG; Babin, 1992) and the fathead minnow, Pimephales promelas (156-kDa VTG; Parks et al, 1999). Based on these findings, further studies are currently in progress.

참고문헌 (20)

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상세보기
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원문보기 상세보기
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상세보기
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Purification, Partial Characterization, and Immunoassay of Vitellogenin from Marbled Sole (Limanda yokohamae) 원문보기

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Purification, Partial Characterization, and Immunoassay of Vitellogenin from Marbled Sole (Limanda yokohamae) 원문보기

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