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

Journal of Medical Virology
Haixia ZhangRuofei Feng

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 ...Continue Reading

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Citations

Jun 27, 2019·Immunological Investigations·Magdalena Bossowska-NowickaLidia Szulc-Dąbrowska
Jan 5, 2021·Frontiers in Oncology·Guang-Quan LiangHenry Wei
May 1, 2021·International Journal of Molecular Sciences·Pharaoh Fellow MwaleYi-Yuan Yang

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