ABSTRACT
OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.
Subject(s)
Avidin/metabolism , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli/metabolism , Membrane Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Avidin/chemistry , Avidin/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Biotin/analogs & derivatives , Biotin/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/cytology , Escherichia coli/genetics , Fluoresceins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Confocal , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood worldwide and Mexico has reported one of the highest incidence rates. An infectious etiology has been suggested and supported by epidemiological evidences; however, the identity of the involved agent(s) is not known. We considered that early transmitted lymphotropic herpes viruses were good candidates, since transforming mechanisms have been described for them and some are already associated with human cancers. In this study we interrogated the direct role of EBV, HCMV, HHV6, and HHV7 human herpes viruses in childhood ALL. Viral genomes were screened in 70 bone marrow samples from ALL patients through standard and a more sensitive nested PCR. Positive samples were detected only by nested PCR indicating a low level of infection. Our result argues that viral genomes were not present in all leukemic cells, and, hence, infection most likely was not part of the initial genetic lesions leading to ALL. The high statistical power of the study suggested that these agents are not involved in the genesis of ALL in Mexican children. Additional analysis showed that detected infections or coinfections were not associated with prognosis.
Subject(s)
Bone Marrow/virology , Mass Screening , Precursor Cell Lymphoblastic Leukemia-Lymphoma/virology , Viruses/isolation & purification , Bone Marrow/pathology , Child , Cytomegalovirus/physiology , Demography , Female , Herpesvirus 4, Human/physiology , Herpesvirus 6, Human/physiology , Herpesvirus 7, Human/physiology , Humans , Limit of Detection , Male , Polymerase Chain Reaction , PrognosisABSTRACT
Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210-239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.
Subject(s)
Antigens, Helminth/immunology , Epitopes/immunology , Trichinella spiralis/immunology , Trichinellosis/prevention & control , Vaccines, Synthetic/immunology , Administration, Intranasal , Animals , Antibodies, Helminth/biosynthesis , Antibodies, Helminth/blood , Antigens, Helminth/genetics , Epitopes/genetics , Genetic Vectors/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Immunoglobulin G/classification , Interferon-gamma/biosynthesis , Interleukin-5/biosynthesis , Intestines/immunology , Intestines/parasitology , Lymph Nodes/cytology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred BALB C , Peyer's Patches/cytology , Peyer's Patches/immunology , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Salmonella typhimurium/genetics , Salmonella typhimurium/immunology , Spleen/cytology , Spleen/immunology , Trichinella spiralis/genetics , Trichinellosis/immunology , Trichinellosis/parasitology , Vaccines, Attenuated , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/geneticsABSTRACT
BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late onset hereditary myopathy of autosomal dominant transmission characterised by ptosis, dysphagia and limb weakness. The disease is caused by short heterozygous expansions of a (GCN)(10) triplet located in the first exon of the PABPN1 gene at chromosome 14q11.1. Most affected individuals from North America and Europe carry a mutant (GCN)(13) allele. Although evidence for a founder mutation effect has been shown in several populations with OPMD, analysis of large groups of patients from different ethnic backgrounds will help to identify the relative contribution of each allele to the disease and a possible genotype-phenotype correlation. METHODS: 22 unrelated patients with OPMD from Mexico, a previously uncharacterised population, were clinically and molecularly analysed. Detailed ophthalmological and clinical examinations were performed in each proband and molecular analysis of the PABPN1 gene was carried out by PCR amplification and allele-specific cloning/sequencing. Two single nucleotide polymorphisms (SNPs) linked to PABPN1 were determined in each individual and in a number of affected first-degree relatives. RESULTS: 15 subjects (68%) carried a mutant (GCN)(15) or (GCG)(11)(GCA)(3)(GCG) PABPN1 allele; the remaining 7 (32%) exhibited an abnormal (GCN)(13) or (GCG)(9)(GCA)(3)(GCG) allele. Analysis of two SNPs linked to PABPN1 strongly suggests that both expanded alleles originate from two independent founder effects. In addition, in this particular population the (GCN)(15) allele was associated with an earlier onset of the disease (mean 46.5 years) compared with the (GCN)(13) allele (mean 54.7 years). CONCLUSION: The results of this study suggest that OPMD in the Mexican population is mostly due to (GCG)(11) or (GCG)(9) PABPN1 expanded alleles arising from two independent founder effect mutations. These findings add to the definition of the genetic features of the disease and to the establishment of a probable genotype-phenotype correlation.