RESUMO
l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.
Assuntos
Asparaginase , Phaseolus , Proteínas Recombinantes , Asparaginase/química , Asparaginase/farmacologia , Asparaginase/genética , Asparaginase/imunologia , Phaseolus/química , Humanos , Cinética , Proteínas Recombinantes/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Leucemia/tratamento farmacológico , Células K562 , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Ensaio de Imunoadsorção Enzimática , Leishmania , Leishmaniose Cutânea , Proteínas de Protozoários , Proteínas Recombinantes , Sensibilidade e Especificidade , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/urina , Proteínas de Protozoários/urina , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/urina , Antígenos de Protozoários/imunologia , Leishmania/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/urina , Adulto , Feminino , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/urina , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Adolescente , Idoso , Urina/química , Urina/parasitologia , Criança , Pré-Escolar , Epitopos de Linfócito B/imunologiaRESUMO
Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 µg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8-12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants.
Assuntos
Hormônio Foliculoestimulante , Animais , Bovinos , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/administração & dosagem , Feminino , Células CHO , Cricetulus , Proteínas Recombinantes , Superovulação/efeitos dos fármacosRESUMO
We conducted a development and standardization of an IgG ELISA assay for serological detection of human orthohantavirus infections using the recombinant antigen rLECH13 produced in bacterial and derived from the LECHV. The evaluation and standardization were carried out by analyzing serum samples from a total of 50 patients with confirmed Hantavirus Pulmonary Syndrome (HPS) diagnosis through the reference technique, 50 negative sera, and 53 patients with other medical conditions. The data from the assay analysis showed a diagnostic sensitivity value of 95% and a diagnostic specificity of 80%. The high sensitivity of this novel assay leads us to conclude that rLECH13 is a feasible option for use in the immunodiagnostic of orthohantavirus infection. Additionally, it is crucial to have an antigen that can be produced under conditions that do not require highly complex laboratories. Furthermore, the new assay is cost-effective, reproducible, and demonstrates excellent performance.
Assuntos
Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Infecções por Hantavirus , Orthohantavírus , Sensibilidade e Especificidade , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , Orthohantavírus/imunologia , Orthohantavírus/genética , Orthohantavírus/isolamento & purificação , Argentina , Infecções por Hantavirus/diagnóstico , Anticorpos Antivirais/sangue , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Imunoglobulina G/sangue , Antígenos ViraisRESUMO
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.
Assuntos
Bacillus subtilis , Celulase , Papel , Proteínas Recombinantes , Águas Residuárias , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/isolamento & purificação , Águas Residuárias/microbiologia , Águas Residuárias/química , Celulase/genética , Celulase/química , Celulase/biossíntese , Celulase/isolamento & purificação , Celulase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Expressão GênicaRESUMO
Acinetobacter baumannii is an opportunistic bacterium that causes infection in several sites. Carbapenem-resistant A. baumannii strains (CRAb) lead the World Health Organization's list of 12 pathogens considered a priority for developing new antimicrobials. The pathogenicity of A. baumannii is related to the different virulence factors employed in the colonization of biotic and abiotic surfaces, biofilm formation and multidrug resistance. We analyze the outer membrane protein FilF from A. baumannii in silico and produce it in recombinant form (rFilF). rFilF protein was successfully expressed in Escherichia coli BL21 Star in an insoluble form. Immunization with rFilF induced significant anti-rFilF IgG antibody production in mice, detected by indirect enzyme-linked immunosorbent assay, since the first evaluation until 49th. On the last experimentation day, the predominant immunoglobulin found was IgG1 followed by IgG2a, IgG2b, IgM, IgG3, and IgA. We observe that interleukins 4 and 10 show significant production after the 28th day of experimentation in mice immunized with rFilF. Anti-rFilF pAbs were able to inhibit biofilm formation in nine CRAb strains evaluated, and in the standard strain ATCC® 19606. These results demonstrate the anti-biofilm activity of anti-rFilF antibodies, promising in the development of a non-antibiotic approach based on the control of CRAb strains.
Assuntos
Acinetobacter baumannii , Anticorpos Antibacterianos , Biofilmes , Carbapenêmicos , Biofilmes/efeitos dos fármacos , Acinetobacter baumannii/imunologia , Acinetobacter baumannii/efeitos dos fármacos , Animais , Anticorpos Antibacterianos/imunologia , Carbapenêmicos/farmacologia , Camundongos , Imunoglobulina G/imunologia , Antibacterianos/farmacologia , Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/genética , Camundongos Endogâmicos BALB C , Feminino , Escherichia coli/genética , Escherichia coli/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genéticaRESUMO
Introduction and objective: p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). Material and methods: This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. Results and discussion: No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.
RESUMO
Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classified in 6 clades and 3 families. We have reported an efficient process for production and purification of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purification was defined after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The final protein purification process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The final purification process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efficient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations. KEY POINTS: ⢠Purification strategy for recombinant PspA1 from Streptococcus pneumoniae ⢠Downstream processing for untagged protein antigens, the case of PspA1 ⢠Purification strategy for PspA variants relies on buried amino acids in their sequences.
Assuntos
Proteínas de Bactérias , Streptococcus pneumoniae , Humanos , Animais , Camundongos , Proteínas de Bactérias/química , Streptococcus pneumoniae/genética , Vacinas Pneumocócicas/metabolismo , Anticorpos Antibacterianos , Camundongos Endogâmicos BALB CRESUMO
Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment.
Assuntos
Antígenos de Protozoários , Leishmaniose Cutânea , Proteínas Recombinantes , Antígenos de Protozoários/imunologia , Humanos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/biossíntese , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/imunologia , Testes Imunológicos/métodosRESUMO
Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential -27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, which favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (~100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (p < 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1ß concentrations, which was associated with an anti-inflammatory effect.