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

J Biochem. 2010 Sep;148(3):335-40. doi: 10.1093/jb/mvq072. Epub 2010 Jun 30.

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 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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Bombyx / genetics*
  • Cloning, Molecular / methods*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Enzyme-Linked Immunosorbent Assay / standards
  • Genetic Vectors
  • Ginsenosides / analysis*
  • Ginsenosides / immunology
  • Nucleopolyhedroviruses / genetics
  • Recombinant Proteins
  • Single-Chain Antibodies / biosynthesis*
  • Single-Chain Antibodies / genetics

Substances

  • Ginsenosides
  • Recombinant Proteins
  • Single-Chain Antibodies
  • ginsenoside Re