Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus

J Med Virol. 2018 Jul;90(7):1184-1191. doi: 10.1002/jmv.25065. Epub 2018 Mar 30.

Abstract

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 μL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.

Keywords: ELISA; SOE-PCR; encephalomyocarditis virus (EMCV); prokaryote expression; qPCR; single-chain variable fragment (scFv).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Viral / biosynthesis
  • Antibodies, Viral / genetics
  • Antibodies, Viral / immunology*
  • Antibodies, Viral / isolation & purification
  • Cardiovirus Infections / prevention & control
  • Cloning, Molecular
  • Disease Models, Animal
  • Encephalomyocarditis virus / immunology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Female
  • Gene Expression
  • Mice, Inbred BALB C
  • Polymerase Chain Reaction
  • Single-Chain Antibodies / biosynthesis
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / isolation & purification
  • Treatment Outcome

Substances

  • Antibodies, Viral
  • Single-Chain Antibodies