Search Results (1,049)

Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes. Full article

Background/objectives. Emerging viral diseases represent an increasing threat to global health security, driven by environmental change, globalization, and intensified human–animal–environment interactions. The COVID-19 pandemic exposed critical weaknesses in preparedness systems but also demonstrated the transformative potential of recombinant vaccine technologies, which enable rapid, scalable, and safe responses to novel pathogens. We aim to examine the role of recombinant vaccine platforms in the management of emerging viral diseases, emphasizing their contribution to health system preparedness and exploring strategies for their integration into preparedness frameworks. Methods. We synthesized the current evidence on recombinant vaccine platforms (viral vector, protein subunit, DNA, and mRNA) through a targeted review of the scientific literature, regulatory documents, and global health policy reports. Drawing from experiences like COVID-19 (mRNA vaccines) and Ebola (rVSV-ZEBOV), we analyzed the advantages, challenges, and lessons from initiatives such as the CEPI, BARDA, HERA, and WHO frameworks. Results. Recombinant vaccine platforms offer significant advantages for epidemic preparedness through rapid adaptability, standardized production, and strong safety profiles. Nonetheless, challenges remain in manufacturing scalability, cold-chain logistics, regulatory harmonization, and equitable global access. Global initiatives such as the CEPI, WHO-led programs, BARDA, and regional manufacturing networks exemplify this collaborative approach, while regulatory mechanisms have proven to be essential to timely vaccine deployment. Conclusions. Recombinant vaccines have redefined preparedness by coupling scientific innovation with operational agility. Strengthening global coordination, regional production capacity, and public trust is essential to ensure that technological progress translates into equitable and effective public health impacts. Full article

The soybean mosaic virus (SMV), a significant viral pathogen impacting soybean cultivation, leads to substantial yield losses and diminishes seed quality. In a prior study, we developed a targeted silencing vector using RNA interference (RNAi) technology targeting the CP gene, which codes for the viral coat proteins in the SMV genome. This vector was delivered into soybean plants through Agrobacterium-mediated transformation. In our current research, we utilized ongoing molecular characterization and resistance screening to identify four genetically pure lines that display moderate to high resistance to SMV. Additionally, the transgenic plants exhibited resistance to three other potyviruses: the bean common mosaic virus, the recombinant soybean mosaic virus, and the watermelon mosaic virus. Greenhouse and field trials conducted with these lines demonstrated that RNAi-mediated silencing of the CP gene significantly enhanced disease resistance. It is noteworthy that, in comparison to the receptor plants, the transgenic plants exhibited no significant differences in maturity, plant height, branching number, node number, pod number, or 100-seed weight. These results offer valuable genetic resources and theoretical support for molecular breeding strategies aimed at combating SMV in soybeans, as well as for RNAi-based methods to control plant viral infections. Full article

The thermophilic bacterium Thermus thermophilus represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from T. thermophilus strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both Escherichia coli and T. thermophilus. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from Thermus spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting T. thermophilus strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications. Full article

The widespread panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 necessitates the development of vaccine platforms capable of rapid adaptation to emerging antigenic variants. Although commercial recombinant turkey herpesvirus (rHVT) vaccines are available, they often utilize heterologous inserts that may fail to optimally limit viral shedding of novel field strains. Here, we report the rapid construction of a homologous rHVT-H5 vaccine expressing the hemagglutinin (HA) gene of a representative clade 2.3.4.4b isolate via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). In vitro characterization confirmed stable HA surface expression and growth kinetics comparable to the parental virus. In specific-pathogen-free (SPF) chickens, rHVT-H5 elicited robust hemagglutination inhibition (HI) antibody titers. Following lethal challenge with a homologous clade 2.3.4.4b H5N1 virus, the vaccine conferred 100% protection against mortality and clinical signs while significantly reduced oropharyngeal sheddings and completely inhibited viral shedding in cloacal samples. These findings demonstrate that an antigenically matched rHVT-H5 constitutes a promising strategy for mitigating the ongoing global threat posed by clade 2.3.4.4b HPAI H5N1. Full article

Background/Objectives: SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette–Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis, other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB (Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods: We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime–boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results: Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime–boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions: Our study highlights the potential of the prime–boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates. Full article

Bovine mastitis remains a globally prevalent disease, with the limitations of antibiotic-based treatments—such as the rise in antimicrobial resistance and the presence of drug residues—highlighting the urgent need for alternative therapeutic approaches. Inflammation is intricately linked to various cytokines and immunomodulatory proteins, among which cyclophilin A (CypA) serves as a pivotal inflammatory mediator, significantly contributing to the initiation and amplification of inflammatory responses under such conditions. The acquisition of high-purity recombinant protein is a fundamental prerequisite for in vitro functional studies of bovine CypA. This study aimed to construct a eukaryotic expression vector for bovine CypA and verify its expression in CHO-K1 cells. Utilizing the bovine CypA gene sequence available in GenBank, the coding region was artificially synthesized and optimized for codon usage, subsequently being inserted into the pPB[Exp] backbone vector via BsrGI and BstEII double digestion. The resulting polycistronic expression vector contained a CAG promoter driving the CypA transcription, an EF1α promoter driving the EGFP reporter gene, a PGK promoter controlling the puromycin resistance gene, and a C-terminal His-tag. Restriction enzyme digestion and bidirectional Sanger sequencing confirmed that the inserted fragment sequence was completely consistent with the optimized design. Robust EGFP fluorescence was observed 24 h post-transfection and remained stable after puromycin selection. qPCR analysis showed that the Ct value of CypA in the experimental group was 16.20 ± 0.04, while no amplification signal was detected in the control group. Additionally, Western blot analysis identified a CypA-specific band at approximately 18 kDa, confirming the correct expression of the exogenous CypA protein in CHO-K1 cells. Collectively, these results demonstrate the successful construction and validation of a bovine CypA eukaryotic expression vector. The established CHO-K1 expression system exhibited stable and efficient expression, thereby providing a robust foundation for future research on the production and application of recombinant CypA protein. Full article

Chymosin is an aspartyl protease widely used in the food industry for milk coagulation during cheesemaking. Although recombinant production has replaced natural extraction from rennet, current heterologous expression systems still face significant challenges, including low solubility, costly purification steps, and enzyme instability after activation. To address these limitations, we sought to develop a more efficient and economical production strategy for bovine chymosin by cloning its codon-optimized prochymosin A gene into Escherichia coli using the pBAD/His vector under the control of the L-arabinose-inducible PBAD promoter. Overexpression of the recombinant gene resulted in the formation of inclusion bodies, which were solubilized with NaOH and refolded by dilution and pH adjustment with glycine. The folded prochymosin was then activated by acidification. To simplify the downstream process and improve enzyme recovery, different immobilization strategies were explored to combine activation, purification, and immobilization in a single step. While polymeric agarose-based supports showed low immobilization efficiency (<20%) due to pore clogging, magnetic nanoparticles completely overcame these limitations, achieving nearly 100% immobilization yield and retaining about 85% of enzymatic activity. This integrated magnetic-based approach provides a cost-effective and scalable alternative for the production and stabilization of active chymosin. Full article

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu²⁺-charged IMAC resin, while moderate binding to Ni²⁺-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production. Full article

The granulation of pomelo (Citrus maxima) juice sacs severely compromises fruit quality and is closely associated with lignin accumulation, a process catalyzed by peroxidases (PODs). Analysis of ‘Sanhong’ pomelo juice sacs collected 175–215 days after flowering revealed that bound peroxidase (BPOD) activity paralleled changes in lignin content, suggesting a potential role for BPOD in lignin biosynthesis. A total of 71 CmPOD genes were identified in the pomelo genome through integrated HMMER and BLAST analyses. Among them, CmPOD52 was selected for functional characterization based on its alkaline peroxidase properties, absence of a CE domain, predicted extracellular localization, and gradually increasing expression pattern revealed by RT-qPCR. Its transient overexpression in ‘Sanhong’ pomelo juice sacs for 36 h increased BPOD activity 2.06-fold (p < 0.01) compared to the empty vector control, indicating that CmPOD52 may be a BPOD gene. The recombinant CmPOD52 protein was expressed in a prokaryotic system, purified, and used in enzymatic assays with sinapyl alcohol as the substrate. The recombinant CmPOD52 protein, assayed at 272 nm with controls (substrate-only blank and heat-inactivated protein), showed an activity of 13.67 ± 0.9 U. The experimental group showed new products, identified by mass spectrometry as sinapyl alcohol dimers, thus suggesting that the recombinant protein catalyzes the dehydrogenation and polymerization of sinapyl alcohol monomers. This study identified CmPOD52, a gene potentially involved in lignin polymerization in pomelo juice sacs, offering a key candidate for further in vivo validation. Full article

Background/Objectives: Inadequate characterization of protective antigens poses a significant challenge to the development of vaccines for African swine fever (ASF), particularly for antigen-dependent formulations such as subunit, mRNA, and recombinant viral vector vaccines. To address this, we aimed to screen African swine fever virus (ASFV) antigens and enhance their immunogenicity using a nanoparticle delivery platform. Methods: Here, six ASFV antigens (p30, p54, pE120R, pH124R, pE184L, and CD2v) were purified and used to immunize pigs individually. The effects of antibodies induced by these six antigens on ASFV replication or hemadsorption was evaluated in primary porcine alveolar macrophages (PAMs). These six antigens were, respectively, conjugated to ferritin via SpyTag/SpyCatcher to prepare six ferritin nanoparticles. A cocktail of the six mixed antigens or a cocktail of the six mixed nanoparticles was used to immunize pigs separately, and the differences in induced humoral and cellular immune responses were compared. Results: Antibodies generated against p30, p54, pE120R, pH124R, and pE184L in immunized pigs significantly inhibited ASFV replication in PAMs, while anti-CD2v antibodies specifically obstructed the hemadsorption of ASFV. Notably, immunization with a cocktail of these antigen-conjugated nanoparticles elicited a stronger virus-inhibitory antibody response compared to immunization with a cocktail of antigen monomers. Furthermore, nanoparticle immunization induced robust cellular immunity, evidenced by elevated serum IFN-γ, increased numbers of ASFV-specific IFN-γ-secreting cells, and an expanded CD8⁺ T cell population. Conclusions: Our study identifies a set of promising ASFV antigen candidates and demonstrates that ferritin nanoparticle delivery synergistically enhances both humoral and cellular immune responses against ASFV, providing a rational strategy for multi-antigen ASF vaccine design. Full article

Plant viruses are useful tools for quickly and easily producing recombinant proteins in plants. Compared to systems that use genetically modified plants, viral vectors are easier to work with and can produce recombinant proteins faster and in larger amounts. Recently, there has been growing interest in using plant viruses as vectors to make vaccines, either as whole proteins or as small parts displayed on plant virus particles. The best examples for this purpose are tobacco mosaic virus, cowpea mosaic virus and potato virus X. Vaccines made using these viruses target various human and animal diseases and have often triggered immune responses and provided protection against infections. This review looks at the benefits of using plant virus vectors, the progress in developing different viral vector systems, and immune studies that support the idea of vaccines made from plant viruses. Full article

Background: Feline panleukopenia virus (FPV) causes acute and frequently fatal disease in cats, underscoring the urgent need for safe, rapidly effective, and scalable vaccines. While virus-like particle (VLP) vaccines are inherently safe and immunogenic, their development is constrained by low yields of recombinant protein in insect cell expression systems. Methods: An optimized baculovirus expression vector system (BEVS) incorporating the hr1-p6.9-p10 transcriptional enhancer and the Ac-ie-01 anti-apoptotic gene was employed to enhance recombinant protein production. VP2 expression levels, viral titers, and hemagglutination activity were quantified using qPCR, SDS-PAGE/Western blotting, transmission electron microscopy (TEM), and functional assays. Immunogenicity and protective efficacy were assessed in both mice and cats through serological analysis, neutralizing antibody detection, and post-challenge clinical monitoring. Results: The optimized BEVS enhanced recombinant protein transcription by 1.5-fold, viral titers by 3.7-fold, and hemagglutination activity by 15-fold. The purified protein self-assembled into uniform 25 nm virus-like particles (VLPs). Immunization elicited earlier responses compared to commercial vaccines. Vaccinated cats maintained normal body temperature, stable leukocyte counts, and minimal viral shedding following FPV challenge. Conclusions: This study validates an enhanced BEVS that effectively overcomes VP2 yield constraints and generates highly immunogenic FPV VLPs. The platform enables rapid-onset protection and offers a scalable strategy for next-generation FPV vaccine development. Full article

Porcine circovirus 2 (PCV2) is a DNA virus that is classified in the genus Circovirus of the Circoviridae family, which is a causative agent of Porcine Circovirus-Associated disease (PCVAD). PCVAD continues to cause substantial losses in global pig farming, with PCV2d being the prevalent genotype worldwide, including in Vietnam. In this study, we focused on generating a recombinant PCV2d Cap protein fused to the GCN4pII motif (Cap2d-pII) in a plant-based system and evaluating its immunogenicity. The Cap2d-pII gene was cloned into a plant expression vector and introduced into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana leaves. Western blot analysis confirmed the high accumulation of the Cap2d-pII protein, which was purified by Immobilized affinity chromatography and used for immunizing mice. ELISA and immunoperoxidase monolayer assay results demonstrated that immunization with the recombinant protein elicited robust humoral and cellular immune responses. At 56 days after immunization, mice vaccinated with the Cap2d-pII protein generated PCV2d-specific IgG titers and IFN-γ responses that were consistent with those in mice receiving the commercial inactivated vaccine. These observations confirm that the plant-expressed Cap2d-pII antigen effectively activates both antibody- and T cell-mediated immune pathways. Collectively, this study identifies the Cap2d-pII protein as a promising plant-derived vaccine candidate for the development of effective and affordable PCV2d subunit vaccines. Full article

The COVID-19 pandemic accelerated vaccine innovation but also exposed weaknesses in global access and manufacturing. Yeast-based platforms, particularly Saccharomyces cerevisiae and Pichia pastoris, also known as Komagataella phaffii, offer a practical complement to vector systems. These eukaryotic microorganisms combine safety, scalability, and cost-effectiveness with the ability to express complex antigens and assemble virus-like particles. Building on the success of the recombinant hepatitis B vaccine, recent advances in glycoengineering, CRISPR-based host optimization, and surface display technologies have expanded the utility of yeast-based platforms for the rapid development of vaccines. Yeast-derived SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines, such as Corbevax and Abdala (CIGB-66), demonstrate that affordable, immunogenic, and thermostable products are feasible at scale. Emerging innovations in glycan humanization, thermostable formulations, and oral or mucosal delivery highlight the potential of yeast-based vaccines for decentralized manufacturing and equitable pandemic preparedness. This review summarizes recent technical and clinical progress in yeast-based vaccine research, positioning these platforms as accessible and adaptable tools for future outbreak responses and global immunization strategies. Full article