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.
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 microg/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.
Sakamoto
Seiichi
S
Department of Pharmacognosy, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Pongkitwitoon
Benyakan
B
Nakamura
Seiko
S
Maenaka
Katsumi
K
Tanaka
Hiroyuki
H
Morimoto
Satoshi
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005822:Genetic Vectors; D036145:Ginsenosides / Q000032:analysis* / Q000276:immunology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics
D036145:Ginsenosides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C049864:ginsenoside Re