RESUMO
Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.
Assuntos
Citometria de Fluxo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Apolipoproteínas E/genética , Corantes Fluorescentes/química , Genoma Humano , Genótipo , Humanos , Microesferas , Oligonucleotídeos/químicaRESUMO
A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Helmintos/imunologia , Glutationa Transferase/imunologia , Biblioteca de Peptídeos , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Bacteriófago M13/genética , Glutationa Transferase/química , Glutationa Transferase/genética , Soros Imunes , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-jun/química , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genéticaRESUMO
A homologue of the mammalian translationally controlled tumor protein (TCTP) was cloned from the human parasite Schistosoma mansoni (SmTCTP). Sequence analysis showed that SmTCTP differed from other reported TCTPs in having only one signature sequence. Subsequently, SmTCTP was cloned in a T7 expression system and expressed as a histidine-tagged fusion protein. Recombinant SmTCTP (rSmTCTP) has a molecular mass of approximately 23 kDa with the histidine tag. Further analysis showed that SmTCTP transcripts and protein are expressed in all life cycle stages of the parasite within the vertebrate hosts. Interestingly, antibodies to SmTCTP were present in the sera of mice 9 weeks after infection with S. mansoni. Characterization studies showed that rSmTCTP is a calcium-binding protein that can cause histamine release from basophil/mast cells and induce eosinophil infiltration. These findings suggest that SmTCTP may have an important role in the development of allergic inflammatory responses associated with schistosomiasis and may be a target for new drug development.
Assuntos
Biomarcadores Tumorais , Proteínas de Ligação ao Cálcio/farmacologia , Proteínas de Helminto/farmacologia , Liberação de Histamina/efeitos dos fármacos , Schistosoma mansoni/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , Relação Dose-Resposta a Droga , Eosinófilos/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/farmacologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Skin-stage schistosomula of Schistosoma mansoni were found to secrete molecules that are pro-apoptotic for skin T lymphocytes as measured by annexin V staining, caspase-3 activity, caspase-8 activities, and DNA fragmentation. Caspase-8 activities in lymphocytes peaked approximately 8 h and caspase-3 activity peaked approximately 16 h after exposure to the parasite secretions. Subset analysis showed that mainly CD4(+) and CD8(+) cells (but not B cells) were susceptible to the parasite-induced pro-apoptotic effect. In situ staining confirmed the presence of apoptotic T cells around challenge parasites in the skin of naive or immunized animals. Analysis of T cells to identify the potential molecular pathway of the parasite-induced apoptosis showed increases in the expression of Fas, FasL, and the Fas-associated death domain. Blocking of FasL with a fusion protein reversed the parasite-induced apoptosis, suggesting a role for the Fas/FasL-mediated pathway in the parasite-induced T cell apoptosis. Subsequent analyses of the secretions of skin-stage schistosomula identified the pro-apoptotic activity as being associated with a protein of approximately 23 kDa. This protein was termed S. mansoni-derived apoptosis-inducing factor.