Trichoderma reesei superoxide dismutase (TrSOD) is a well characterized enzyme being stable between 30 and 90°C for 1 h with activity at pH between 2.6 and 9.0. This work aimed to clone, express, purify and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and Abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein) and Ab-TrSOD were expressed in E.coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3 and 6 μmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide (tBH) and evaluated for ROS generation, in the presence or not of the recombinant enzymes.
TAT-TrSOD-Yfp was able to decrease the generation of ROS in 15% when used in the concentrations of 3 and 6 μmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19 and 14% at the concentrations of 1 and 6 μmol/L, respectively, indicating that this joplinks Recombinant Human Regulator of G-protein form was more effective in reducing oxidative stress compared to SOD without the cell penetrating peptide (CPP). Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development of products for antioxidant protection of the skin that may have therapeutic and cosmetic application. This article is protected by copyright.
Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems
The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model.
An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.
Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips
The coronavirus disease 2019 (COVID-19) are outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of Severe Acute Respiratory syndrome Coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drag target.
Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in E.coli and obtained 15 mAbs against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established.
In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen. This article is protected by copyright. All rights reserved.
Direct enzyme-linked aptamer assay (DELAA) for diagnosis of toxoplasmosis by detection of SAG1 protein in mice and humans
Toxoplasma gondii is a single-celled parasite commonly found in mammals and birds. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key molecule for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology. Recombinant SAG1 (r-SAG1) of Toxoplasma WH3 strain (type Chinese 1) was expressed in E.coli and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1 antigen, with systematic evolution of ligands by exponential enrichment (SELEX) strategy.
The specific aptamers were screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 (n-SAG1) obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified for Toxoplasma DNAs by PCR amplification. As results, the soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, followed by biotinylation. The selected aptamers were amplified by PCR and DNA sequencing.
The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals with minimal difference in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for T.gondii circulating antigen test. We concluded that a new direct enzyme-linked aptamer assay (DELAA) was developed for the detection of the n-SAG1 protein of T. gondii. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered an efficient method for the diagnosis of active as well as reactivated toxoplasmosis.
Preparation and identification of rat polyclonal antibody against SARS-CoV-2 main protease (Mpro)
Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column.
The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.