RESUMO
The complete gag gene from small ruminant lentiviruses (SRLV) encodes for a polyprotein of 55 kDa, known as p55gag. p55gag presents multiple antigenic epitopes, which can be recognized by antibodies, increasing the opportunity to detect SRLV-positive animals. Therefore, this polyprotein is considered an excellent candidate to use in diagnostic tests to detect antibodies against SRLV. Different studies have suggested that the selection of the recombinant antigen, which must be representative of the virus strains circulating in the test population, is crucial to avoid false negative results. Thus, the use of proteins from different viral strains isolated from goats or sheep of a given region or country may be a useful strategy to increase the ability to detect SRLV-infected animals. In the present study, the pMAL-p5X vector was used to express and purify p55gag (now called rp55gag for recombinant polyprotein 55 gag). The cloned gene was inserted downstream from the malE gene of Escherichia coli, which encodes a maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein. The complete gag gene was amplified by RT-PCR. Finally, after digestion, the product was cloned into the pMAL-p5X vector and used to transform E. coli ER2325 cells. After the purification of MBP-rp55gag by affinity chromatography, the eluted fraction was observed by SDS-PAGE and Western Blot (WB). The WB was carried out with 85 serum samples from small ruminants previously analysed and compared by two commercial ELISAs. The results show that 76 of the serum samples were concordant with those by both ELISAs. Regarding the other nine serum samples, which showed discordant results between both ELISAs, were positive by WB. The results thus show that the rp55gag could be considered as an antigen in a confirmatory diagnostic assay to detect SRLV by WB. For this purpose, a future study with a high number of sera to determine the test specificity and sensitivity, using the p55gag of the circulating strain in Argentina will be necessary.
Assuntos
Doenças das Cabras , Infecções por Lentivirus , Doenças dos Ovinos , Animais , Escherichia coli , Doenças das Cabras/diagnóstico , Cabras , Lentivirus/genética , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/veterinária , Proteínas Ligantes de Maltose/genética , Filogenia , Poliproteínas/genética , Ruminantes , Ovinos , Doenças dos Ovinos/diagnósticoRESUMO
Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3â¢60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3â¢60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of kcat 1â¯min-1 and Km of 34⯵M.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Cinética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , TermodinâmicaRESUMO
A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anemia Infecciosa Equina/virologia , Imunodifusão/métodos , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Proteínas Ligantes de Maltose/análise , Proteínas do Core Viral/análise , Animais , Ensaio de Imunoadsorção Enzimática/instrumentação , Anemia Infecciosa Equina/diagnóstico , Anemia Infecciosa Equina/imunologia , Cavalos , Imunodifusão/instrumentação , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
BACKGROUND: Stimulation of Toll-like receptors (TLRs) by microbial products has been utilised to potentiate immune responses against haematologic malignancies. The maltose-binding protein (MBP) of Escherichia coli could induce the activation of immune cells via TLR4. The aim of the present study was to investigate whether TLRs mediated the biological effects of MBP on U937 and Jurkat cells in vitro. METHODS We observed the effect of MBP on U937 and Jurkat cells by using the WST, cell cycle analysis and morphological observation. Further, cells were stimulated with MBP for indicated times and doses, and detected by RT-PCR, western blotting, immunohistochemistry and immunofluorescence staining to investigate the mechanisms involved in cell viability. RESULTS: MBP enhanced the viability of U937 and Jurkat cells, and the effects were blocked by anti-TLR2, but not anti-TLR4 in U937 cells. Further studies confirmed that MBP was able to directly bind to U937 and Jurkat cells and modulate TLR expression. The effects of MBP depended on the activation of NF-κB and MAP kinase in U937 and Jurkat cells. CONCLUSIONS: Our results demonstrated that MBP could directly promote U937 cell viability via TLR2. It suggested that MBP may be used as an adjuvant for participating in the immunotherapy of haematologic malignancies.
Assuntos
Diferenciação Celular , Proliferação de Células , Proteínas Ligantes de Maltose/metabolismo , Receptor 2 Toll-Like/metabolismo , Western Blotting , Ciclo Celular , Escherichia coli , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Células Jurkat , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/isolamento & purificação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células U937 , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bacteria use two-component signal transduction systems to detect and respond to environmental changes. These systems have been studied systematically in Escherichia coli as a model organism. Most of the signal transduction systems present in E. coli are conserved in related pathogenic bacteria; however, differences in regulation by these systems have been reported from one bacterial species to another [Oropeza, R., and Calva, E. (2009). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol. Lett.292, 282-290]. Our laboratory has been interested in studying the OmpR/EnvZ two-component system in S. enterica. In S. enterica serovar Typhi (Typhi), it regulates the expression of the porin genes, namely ompC, ompF, ompS1, and ompS2. OmpR proteins are identical between E. coli and Typhi, but several differences exist between the EnvZ proteins. To define whether some differences in porin regulation are due to changes on EnvZ, we decided to overexpress and purify E. coli, Typhi, and S. enterica serovar Typhimurium (Typhimurium) EnvZ proteins fused to the maltose-binding protein (MBP) as a purification tag. Differences in the autophosphorylation level of these proteins were evidenced. Hence, considering the differences at the amino acid level between E. coli and Typhi EnvZ proteins, several mutations were introduced in the Typhi EnvZ protein in order to try to find the amino acids affecting the enzymatic activity of the protein. We found that Cys354 plays an important role in defining the enzymatic activity of this histidine kinase. Here, we report the automated purification of a collection of MBP-EnvZ fusions using a mini-chromatography commercial system, but adapting an amylose affinity column packed by ourselves.