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Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Atzingen MV, Rodriguez D, Siqueira GH, Leite LC, Nascimento AL - Biomed Res Int (2014)

Bottom Line: Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity.Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins.The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil.

ABSTRACT
Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

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(a) Schematic representation of the expression cassettes. The genetically fused genes were obtained from individually cloned genes in pAE vector, and, then, surface protein genes were digested at PvuII/NcoI restriction sites and ligated in the same sites into pAE-DnaK construct. Depicted are T7 phage RNA polymerase promoter, ribosome-binding site (RBS), ATG start códon, 6X Histidine tag, restriction cloning sites, and the 2X (Gly-Pro) flexible hinge. (b) Analysis of purified recombinant proteins by SDS-PAGE. Purified recombinant protein eluted from Ni+2-charged Sepharose column with 1 M imidazole are visualized by Coomassie blue staining. Lane H (HMW) and L (LMW): high and low molecular mass protein markers; In kDa: lane 1: DnaK (68.9); lane 2: rLIC10494 (25.1); lane 3: DnaK-rLIC10494 (92.5); lane 4: rLIC12730 (75.7); lane 5: DnaK-rLIC12730 (143.1); lane 6: Lsa21 (19.8); lane 7: DnaK-Lsa21 (87.2); lane 8: Lp95 C-terminal (43.6); lane 9: DnaK-Lp95 C-terminal (110.9).
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fig1: (a) Schematic representation of the expression cassettes. The genetically fused genes were obtained from individually cloned genes in pAE vector, and, then, surface protein genes were digested at PvuII/NcoI restriction sites and ligated in the same sites into pAE-DnaK construct. Depicted are T7 phage RNA polymerase promoter, ribosome-binding site (RBS), ATG start códon, 6X Histidine tag, restriction cloning sites, and the 2X (Gly-Pro) flexible hinge. (b) Analysis of purified recombinant proteins by SDS-PAGE. Purified recombinant protein eluted from Ni+2-charged Sepharose column with 1 M imidazole are visualized by Coomassie blue staining. Lane H (HMW) and L (LMW): high and low molecular mass protein markers; In kDa: lane 1: DnaK (68.9); lane 2: rLIC10494 (25.1); lane 3: DnaK-rLIC10494 (92.5); lane 4: rLIC12730 (75.7); lane 5: DnaK-rLIC12730 (143.1); lane 6: Lsa21 (19.8); lane 7: DnaK-Lsa21 (87.2); lane 8: Lp95 C-terminal (43.6); lane 9: DnaK-Lp95 C-terminal (110.9).

Mentions: The genes, DnaK, LIC10368, LIC10494, LIC12730, and LIC12690, were amplified from L. interrogans serovar Copenhageni genomic DNA with complementary primers, designed with the restriction sequences at forward and reverse directions. Table 1 summarizes gene locus, recombinant protein given name, NCBI reference number, genome annotated protein function, sequence of the primers with the restriction cloning sites, and predicted molecular mass of the individual recombinant protein. The genes were first individually cloned into pAE plasmid. The construction generated with pAE-DnaK allows the directional cloning of other guest DNA inserts in fusion at the carboxy-terminus of DnaK gene. The fused proteins have 6X His tag at N-terminus of DnaK and a flexible 2X (Gly-Pro) hinge region incorporated between the C-terminus of DnaK and the N-terminus of the selected gene. The hinge purposes to separate the two components of the protein fusion, thus allowing each to undergo folding without steric hindrance from the other [22]. The scheme of the expression cassettes of the constructs is depicted in Figure 1(a).


Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Atzingen MV, Rodriguez D, Siqueira GH, Leite LC, Nascimento AL - Biomed Res Int (2014)

(a) Schematic representation of the expression cassettes. The genetically fused genes were obtained from individually cloned genes in pAE vector, and, then, surface protein genes were digested at PvuII/NcoI restriction sites and ligated in the same sites into pAE-DnaK construct. Depicted are T7 phage RNA polymerase promoter, ribosome-binding site (RBS), ATG start códon, 6X Histidine tag, restriction cloning sites, and the 2X (Gly-Pro) flexible hinge. (b) Analysis of purified recombinant proteins by SDS-PAGE. Purified recombinant protein eluted from Ni+2-charged Sepharose column with 1 M imidazole are visualized by Coomassie blue staining. Lane H (HMW) and L (LMW): high and low molecular mass protein markers; In kDa: lane 1: DnaK (68.9); lane 2: rLIC10494 (25.1); lane 3: DnaK-rLIC10494 (92.5); lane 4: rLIC12730 (75.7); lane 5: DnaK-rLIC12730 (143.1); lane 6: Lsa21 (19.8); lane 7: DnaK-Lsa21 (87.2); lane 8: Lp95 C-terminal (43.6); lane 9: DnaK-Lp95 C-terminal (110.9).
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Related In: Results  -  Collection

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fig1: (a) Schematic representation of the expression cassettes. The genetically fused genes were obtained from individually cloned genes in pAE vector, and, then, surface protein genes were digested at PvuII/NcoI restriction sites and ligated in the same sites into pAE-DnaK construct. Depicted are T7 phage RNA polymerase promoter, ribosome-binding site (RBS), ATG start códon, 6X Histidine tag, restriction cloning sites, and the 2X (Gly-Pro) flexible hinge. (b) Analysis of purified recombinant proteins by SDS-PAGE. Purified recombinant protein eluted from Ni+2-charged Sepharose column with 1 M imidazole are visualized by Coomassie blue staining. Lane H (HMW) and L (LMW): high and low molecular mass protein markers; In kDa: lane 1: DnaK (68.9); lane 2: rLIC10494 (25.1); lane 3: DnaK-rLIC10494 (92.5); lane 4: rLIC12730 (75.7); lane 5: DnaK-rLIC12730 (143.1); lane 6: Lsa21 (19.8); lane 7: DnaK-Lsa21 (87.2); lane 8: Lp95 C-terminal (43.6); lane 9: DnaK-Lp95 C-terminal (110.9).
Mentions: The genes, DnaK, LIC10368, LIC10494, LIC12730, and LIC12690, were amplified from L. interrogans serovar Copenhageni genomic DNA with complementary primers, designed with the restriction sequences at forward and reverse directions. Table 1 summarizes gene locus, recombinant protein given name, NCBI reference number, genome annotated protein function, sequence of the primers with the restriction cloning sites, and predicted molecular mass of the individual recombinant protein. The genes were first individually cloned into pAE plasmid. The construction generated with pAE-DnaK allows the directional cloning of other guest DNA inserts in fusion at the carboxy-terminus of DnaK gene. The fused proteins have 6X His tag at N-terminus of DnaK and a flexible 2X (Gly-Pro) hinge region incorporated between the C-terminus of DnaK and the N-terminus of the selected gene. The hinge purposes to separate the two components of the protein fusion, thus allowing each to undergo folding without steric hindrance from the other [22]. The scheme of the expression cassettes of the constructs is depicted in Figure 1(a).

Bottom Line: Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity.Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins.The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil.

ABSTRACT
Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

Show MeSH
Related in: MedlinePlus