The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach uses SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDT’s). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.
Cytoreg is an ionic therapeutic agent comprising a mixture of hydrochloric, sulfuric, phosphoric, hydrofluoric, oxalic, and citric acids. In diluted form, it has demonstrated efficacy against human cancers in vitro and in vivo. Although Cytoreg is well tolerated in mice, rats, rabbits, and dogs by oral and intravenous administration, its mechanism of action is not understood. The acidic nature of Cytoreg could potentially disrupt the pH and levels of ions and dissolved gases in the blood. Here, we report the effects of the intravenous administration of Cytoreg on the arterial pH, oxygen and carbon dioxide pressures, and bicarbonate, sodium, potassium, and chloride concentrations. Our results demonstrate that Cytoreg increases oxygen levels and can be used from a therapeutic point of view.
Cytoreg is an ionic therapeutic agent comprising a mixture of hydrochloric, sulfuric, phosphoric, hydrofluoric, oxalic, and citric acids. In diluted form, it has demonstrated efficacy against human cancers in vitro and in vivo . Although Cytoreg is well tolerated in mice, rats, rabbits, and dogs by oral and intravenous administration, its mechanism of action is not documented. The acidic nature of Cytoreg could potentially disrupt the pH and levels of ions and dissolved gases in the blood. Here, we report the effects of the intravenous administration of Cytoreg on the arterial pH, oxygen and carbon dioxide pressures, and bicarbonate, sodium, potassium, and chloride concentrations. Our results demonstrate that Cytoreg does not disturb the normal blood pH, ion levels, or carbon dioxide content, but increases oxygen levels in rats. These data are consistent with the excellent tolerability of intravenous Cytoreg observed in rabbits, and dogs. The study was approved by the Bioethics Committee of the University of the Andes, Venezuela (CEBIOULA) (approval No. 125) on November 3, 2019.
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