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

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) -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 10 TCID 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.

Design and preliminary application of affinity peptide based on the structure of the porcine circovirus type II Capsid (PCV2 Cap)

“…The specificity of these five peptides was evaluated by P/N values and cross-reactivity. When the P/N value is greater than 2.1, the specificity of the peptide is good (Zhang et al, 2018). Figure 3A shows that the five peptides for Cap had P/N values greater than 2.1.…”

Encephalomyocarditis virus in a capybara (Hydrochoerus hydrochaeris)

“…17,46,47 Administration of antibodies and a DNA vaccine have also been reported to provide effective protection in mice, and a genetically engineered Mengo virus vaccine has been reported to provide immunity in baboons, macaques and domestic pigs. [48][49][50] Variation in immune response after vaccination is common, and factors such as antigen concentration, viral strain, adjuvant and host immunocompetence may affect immunity. 51 Naturally acquired antibody titres do not appear to persist despite endemic infection, and high titres are not necessarily protective.…”

“…Inactivated vaccines against EMCV have been shown to be effective in mice, elephants and pigs, Barbary sheep ( Ammotragus lervia ), Indian Antelope ( Antilope cervicapra ), Eastern wallaroos ( Macropus robustus ), chimpanzees and Rhesus macaques ( Macaca mulatta ) 17,46,47 . Administration of antibodies and a DNA vaccine have also been reported to provide effective protection in mice, and a genetically engineered Mengo virus vaccine has been reported to provide immunity in baboons, macaques and domestic pigs 48–50 . Variation in immune response after vaccination is common, and factors such as antigen concentration, viral strain, adjuvant and host immunocompetence may affect immunity 51 .…”

Anti-CXCR4 Single-Chain Variable Fragment Antibodies Have Anti-Tumor Activity

“…It contains no antigenicity if it is isolated from a human scFv library. It can be produced in bacteria, thereby greatly reducing the manufacturing cost (23,24). scFv does not contain Fc domain of mAb so that it cannot mediate antibody dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-mediated opsonization.…”