Title Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin /
Authors Dapkūnas, Žilvinas ; Baranauskas, Aurimas ; Mickienė, Gitana ; Plečkaitytė, Milda ; Žvirblis, Gintautas
DOI 10.1016/j.ejbt.2017.05.003
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Is Part of Electronic journal of biotechnology.. Valparaíso : Pontificia Universidad Católica de Valparaíso. 2017, Vol. 28, p. 52-57.. ISSN 0717-3458
Keywords [eng] Circulation half-life ; ELISA ; Gardnerella vaginalis ; Genetic fusion ; Hemolysis ; Medical biotechnology ; Pore-forming toxin ; Recombinant antibodies ; Therapeutic agent ; Vaginal bacterium ; Virulence factor
Abstract [eng] Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.
Published Valparaíso : Pontificia Universidad Católica de Valparaíso
Type Journal article
Language English
Publication date 2017