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Development and partial characterisation of an antiserum against apolipoprotein B of the short-finned eel, Anguilla australis

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Abstract

Despite its key role in transportation of triacylglycerides in blood, the distribution, localisation and molecular weight variants of apolipoprotein B (Apob) in teleost fish have essentially escaped study. To address this, a specific short-finned eel (Anguilla australis) Apob antiserum was produced by an immunised rabbit, purified and partially characterised. Localisation of Apob at both the mRNA (in situ hybridisation) and protein (immunohistochemistry) levels mirrored that of mammals; thus immunostaining was confined to the interstitial spaces of the liver and the vascular core of the intestinal villi. Immunostaining of proteins by Western blotting, followed by high-resolution LC–MS, indicated that peptide sequence coverage of Apob in low-density lipoproteins spanned the full-length protein. We conclude that only full-length Apob is produced by eels and that both liver and intestine are key sites for its synthesis.

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Abbreviations

2ME:

2-Mercaptoethanol

Apob:

Apolipoprotein B

BCIP:

5-Bromo-4-chloro-3-indolyl phosphate

CBB:

Coomassie brilliant blue

DEPC:

Diethylpyrocarbonate

DIG:

Digoxigenin

Eef1α:

Eel elongation factor one alpha

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LC–MS:

Liquid chromatography–mass spectrometry

Ldl:

Low-density lipoprotein

NBT:

4-Nitro blue tetrazolium chloride

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

PVDF:

Polyvinylidene fluoride

qPCR:

Quantitative polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

tag:

Triacylglyceride

Vldl:

Very low-density lipoprotein

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Acknowledgments

The authors would like to thank Mr Ali Falahati (Department of Zoology, University of Otago) and Miss Shuang Li (Faculty of Fisheries Sciences, Hokkaido University) for help with recombinant protein purification and the Division of Science (University of Otago) for the Grant-in-Aid, Postgraduate Travel Grant allowing for travel to Japan. We also acknowledge the financial support from a PBRF Research Enhancement Grant (2013_ML63) from the Department of Zoology, University of Otago (to PML).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experimental protocol was approved by the Institutional Animal Care and Use Committee of the National University Corporation, Hokkaido University and the animal experiment was performed in accordance with the Hokkaido University manual for implementing animal experimentation.

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Authors and Affiliations

  1. Department of Zoology, University of Otago, 340 Great King Street, PO Box 56, Dunedin, 9054, New Zealand

    Erin L. Damsteegt, Yuichi Ozaki & P. Mark Lokman

  2. Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido, 041-8611, Japan

    Hiroko Mizuta, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara, Shigeho Ijiri & Shinji Adachi

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  1. Erin L. Damsteegt
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  2. Hiroko Mizuta
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  3. Yuichi Ozaki
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Correspondence to Erin L. Damsteegt.

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Communicated by I. D. Hume.

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Damsteegt, E.L., Mizuta, H., Ozaki, Y. et al. Development and partial characterisation of an antiserum against apolipoprotein B of the short-finned eel, Anguilla australis . J Comp Physiol B 184, 589–599 (2014). https://doi.org/10.1007/s00360-014-0821-4

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