RESUMO
A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.
Assuntos
Escherichia coli , Streptococcus pneumoniae , Streptococcus pneumoniae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Estreptolisinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.
Assuntos
Streptococcus pneumoniae , Estreptolisinas , Receptor 4 Toll-Like , Camundongos , Animais , Streptococcus pneumoniae/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/metabolismo , Células DendríticasRESUMO
Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized intraperitoneally with recombinant pneumolysin sorbed on Al(OH)3 (200 µg per mouse). In 2 weeks after immunization, the isotypes of antibodies to recombinant pneumolysin in the serum of immunized mice were determined by ELISA. The animals were infected with Streptococcus pneumoniae serotype 3. Immunization with recombinant pneumolysin induced the production of anti-pneumolysin antibodies, mainly of IgG1 subisotype. On day 21 after intraperitoneal infection with S. pneumoniae serotype 3 in a dose of 106 microbial cells, the survival rate of animals immunized with recombinant pneumolysin in a dose of 25 µg/mouse was 67% vs. 0% in the control (p<0.001). Recombinant pneumolysin could be considered as a promising protective antigen for inclusion in the serotype-independent vaccine against S. pneumoniae.
