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CLONAMIENTO, EXPRESIÓN Y SEROREACTIVIDAD DE LA PROTEÍNA RECOMBINANTE DE ENSAMBLAJE DE LIPOPOLISACÁRIDOS -- D (LptD) DE Bartonella bacilliformis.

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Title: CLONAMIENTO, EXPRESIÓN Y SEROREACTIVIDAD DE LA PROTEÍNA RECOMBINANTE DE ENSAMBLAJE DE LIPOPOLISACÁRIDOS -- D (LptD) DE Bartonella bacilliformis.
Alternate Title: CLONING, EXPRESSION AND SEROREACTIVITY OF THE RECOMBINANT LIPOPOLYSACCHARIDE ASSEMBLY PROTEIN -- D (LptD) FROM Bartonella bacilliformis.
Authors: Flores-Nuñez, Astrid1,2 astridflo44@gmail.com, Ventura, Gladis1, Bailon, Henri1, Marcelo, Adolfo3, Sandoval, Gustavo2, Padilla-Rojas, Carlos1
Source: Revista Peruana de Medicina Experimental y Salud Pública. 2022, Vol. 39 Issue 1, p15-23. 9p.
Abstract (English): 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 °C, 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. [ABSTRACT FROM AUTHOR]
Abstract (Spanish): Objetivo. Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). Materiales y métodos. Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-β-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. Resultados: In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 °C, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. Conclusiones. El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión. [ABSTRACT FROM AUTHOR]
Copyright of Revista Peruana de Medicina Experimental y Salud Pública is the property of Instituto Nacional de Salud (Peru) and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: CLONAMIENTO, EXPRESIÓN Y SEROREACTIVIDAD DE LA PROTEÍNA RECOMBINANTE DE ENSAMBLAJE DE LIPOPOLISACÁRIDOS -- D (LptD) DE Bartonella bacilliformis.
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  Data: CLONING, EXPRESSION AND SEROREACTIVITY OF THE RECOMBINANT LIPOPOLYSACCHARIDE ASSEMBLY PROTEIN -- D (LptD) FROM Bartonella bacilliformis.
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  Data: <searchLink fieldCode="AR" term="%22Flores-Nuñez%2C+Astrid%22">Flores-Nuñez, Astrid</searchLink><relatesTo>1,2</relatesTo><i> astridflo44@gmail.com</i><br /><searchLink fieldCode="AR" term="%22Ventura%2C+Gladis%22">Ventura, Gladis</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Bailon%2C+Henri%22">Bailon, Henri</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Marcelo%2C+Adolfo%22">Marcelo, Adolfo</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Sandoval%2C+Gustavo%22">Sandoval, Gustavo</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Padilla-Rojas%2C+Carlos%22">Padilla-Rojas, Carlos</searchLink><relatesTo>1</relatesTo>
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  Data: <searchLink fieldCode="JN" term="%22Revista+Peruana+de+Medicina+Experimental+y+Salud+Pública%22">Revista Peruana de Medicina Experimental y Salud Pública</searchLink>. 2022, Vol. 39 Issue 1, p15-23. 9p.
– Name: Abstract
  Label: Abstract (English)
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  Data: 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 °C, 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. [ABSTRACT FROM AUTHOR]
– Name: Abstract
  Label: Abstract (Spanish)
  Group: Ab
  Data: Objetivo. Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). Materiales y métodos. Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-β-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. Resultados: In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 °C, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. Conclusiones. El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
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  Data: <i>Copyright of Revista Peruana de Medicina Experimental y Salud Pública is the property of Instituto Nacional de Salud (Peru) and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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