RESUMO
Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , Sequência de Aminoácidos , COVID-19/sangue , COVID-19/imunologia , Teste Sorológico para COVID-19/métodos , Estudos de Casos e Controles , Clonagem Molecular , Proteínas do Nucleocapsídeo de Coronavírus/genética , Ensaio de Imunoadsorção Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Soros Imunes/química , Imunoglobulina M/sangue , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
Helminth-derived products, either released into the circulation during the course of the infection or isolated after in vitro cultivation of the parasite and applied by the injection, are able to suppress the host immune response to autoantigens and allergens, but mechanisms could differ. Prophylactic application of Trichinella spiralis excretory-secretory muscle larvae (ES L1) products ameliorates experimental autoimmune encephalomyelitis (EAE) with the same success as infection did. However, a shift to the Th2-type response in the periphery and in the central nervous system, accompanied by activation of regulatory mechanisms, had a striking, new feature of increased proportion of unconventional CD4(+)CD25(-)Foxp3(+) regulatory cells both in the periphery and in the central nervous system of animals treated with ES L1 before the induction of EAE.
Assuntos
Antígenos de Helmintos/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/terapia , Proteínas de Helminto/imunologia , Linfócitos T Reguladores/imunologia , Trichinella spiralis/imunologia , Animais , Antígenos CD4/metabolismo , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunomodulação , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ratos , Ratos WistarRESUMO
In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.
