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Revista Peruana de Medicina Experimental y Salud Publica
versão impressa ISSN 1726-4634
Resumo
FLORES-NUNEZ, Astrid et al. Cloning, expression and seroreactivity of the recombinant lipopolysaccharide assembly protein - D (LptD) from Bartonella bacilliformis. Rev. perú. med. exp. salud publica [online]. 2022, vol.39, n.1, pp.15-23. Epub 31-Mar-2022. ISSN 1726-4634. http://dx.doi.org/10.17843/rpmesp.2022.391.9292.
Objective.
To evaluate in silico and at the serological level the antigenic potential of the recombinant extracellular domain of the lipopolysaccharide assembly protein - D (LptD) of Bartonella bacilliformis (dexr_LptD).
Materials and Methods.
Through in silico analysis, we selected a B. bacilliformis protein with antigenic and immunogenic potential. The selected protein gene was cloned into Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) pLysS. Recombinant protein was expressed using isopropyl-β-D-1-thiogalactopyranoside (IPTG) and induction conditions were optimized. Finally, it was purified with Ni-IDA resin (His60 Ni Superflow) and a Western Blot assay was conducted.
Results.
In silico, the selected protein was LptD because it is located in the outer membrane and is antigenic and immunogenic. Optimized conditions for dexr_LptD induction were 0.5 mM IPTG, 16 hours, TB (Terrific Broth) medium, 3% (v/v) ethanol, 28 ºC, OD600: 1-1.5 and 200 rpm. Purification was carried out under denaturating conditions on a small scale and we obtained 2.6 μg/mL of partially purified dexr_LptD. The Western Blot assay showed a positive reaction between the sera from patients with Carrión's Disease and dexr_LptD, which shows the antigenicity of dexr_LptD.
Conclusions.
The dexr_LptD shows antigenicity both in silico and at the serological level, these results are the basis for further studies on vaccine candidates against Carrion’s Disease.
Palavras-chave : Bartonella infections; LptD protein; Bartonella bacilliformis; Recombinant proteins; Vaccine Antigenicity; Computational biology.