Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides

Seiichi Sakamoto, Benyakan Pongkitwitoon, Seiko Nakamura, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

研究成果: ジャーナルへの寄稿記事

6 引用 (Scopus)

抄録

A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 μg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.

元の言語英語
ページ(範囲)335-340
ページ数6
ジャーナルJournal of biochemistry
148
発行部数3
DOI
出版物ステータス出版済み - 9 1 2010

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Nucleopolyhedrovirus
Ginsenosides
Single-Chain Antibodies
Immunosorbents
Bombyx
Quality Control
Quality control
Assays
Enzyme-Linked Immunosorbent Assay
Escherichia coli
DNA
Larva
Enzymes
Monoclonal Antibodies
Hemolymph
Melitten
Affinity chromatography
Ion chromatography
Protein Sorting Signals
Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

これを引用

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abstract = "A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 μg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.",
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AU - Sakamoto, Seiichi

AU - Pongkitwitoon, Benyakan

AU - Nakamura, Seiko

AU - Maenaka, Katsumi

AU - Tanaka, Hiroyuki

AU - Morimoto, Satoshi

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