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Construction, Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae

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Abstract

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly4Ser)3 between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

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Abbreviations

ABTS 2:

2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt

BmNPV:

Bombyx mori nucleopolyhedrovirus

bp:

Base pairs

CR:

Cross-reactivities

2,4-D:

2,4-Dichlorophenoxyacetic acid

2,4-D-scFv:

Single-chain variable fragment antibody against 2,4-D

ELISA:

Enzyme-linked immunosorbent assay

HMSS:

Honeybee melittin signal sequence

HRP:

Horseradish peroxidase

K D :

Dissociation constant

PBS:

Phosphate-buffered saline

scFv:

Single-chain variable fragment

VH:

Heavy chain variable region

VL:

Light-chain variable region

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Acknowledgments

The research in this paper was supported, in part, by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This work was also funded by a Grant in Aid from the Japan Society for the Promotion of Science Asian CORE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Author notes

  1. Katsumi Maenaka

    Present address: Department of Biomolecular Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-12, W-6, Kita-ku, Sapporo, 060-0812, Japan

Authors and Affiliations

  1. Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Seiichi Sakamoto, Benyakan Pongkitwitoon, Kaori Sasaki-Tabata, Yusuke Tanizaki, Hiroyuki Tanaka & Satoshi Morimoto

  2. Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Seiko Nakamura

  3. Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Katsumi Maenaka

Authors
  1. Seiichi Sakamoto
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Correspondence to Hiroyuki Tanaka.

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Sakamoto, S., Pongkitwitoon, B., Nakamura, S. et al. Construction, Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae. Appl Biochem Biotechnol 164, 715–728 (2011). https://doi.org/10.1007/s12010-011-9168-4

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