RESUMO
Zika virus became a major public health problem in early 2015, when cases of Guillain-Barré syndrome and microcephaly were associated with viral infection. Currently, ZIKV is endemic in all tropical areas of the world, and the chance for future Zika epidemics remains very real and accurate diagnosis is crucial. The aim of this work was to select specific ssDNA aptamers that bind to the entire Zika virus and can be used to compose specific diagnostics, without cross-reactivity with other flaviviruses. Zika virus was cultivated in Vero cells and used as a target for aptamer selection. Aptamers specific for the ZIKV were selected using whole-virus SELEX, with counterselection for other flavivirus. Secondary and tertiary structures were evaluated and the molecular anchoring between the aptamers and target were simulated by the HDOCK server. Aptamer interaction was evaluated by ELISA/ELASA and the dissociation constant (Kd) was calculated by thermophoresis. Four ZIKV-specific aptamers were selected. The best two were further characterized and proved to be specific for ZIKV. Aptamers are capable of binding specifically to the ZIKV and differentiate from Dengue virus. The aptamers selected in this work can be used as capture agents in the composition of diagnostic tests to specifically detect ZIKV infection.
Assuntos
Flavivirus , Infecção por Zika virus , Zika virus , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Reações Cruzadas , DNA de Cadeia Simples , Humanos , Células VeroRESUMO
l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of â¼38â¯kDa. The native enzyme was a tetramer with a molecular mass of approximately 178â¯kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.
Assuntos
Antineoplásicos/metabolismo , Asparaginase/genética , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Antineoplásicos/química , Asparaginase/química , Asparaginase/metabolismo , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Expressão Gênica , Glutamina/metabolismo , Humanos , Peso Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.