{"title":"Purification and characterization of kyasanur forest disease virus EDIII domain of major envelope glycoprotein.","authors":"Manjima Das, Vivek Kumar, Rishav Madhukalya, Rohit Gupta, Vidushi Agarwal, Shweta Choudhary, Mandar Bhutkar, Shailly Tomar, Dilip Kumar, Rajesh Kumar","doi":"10.1016/j.jviromet.2024.115089","DOIUrl":null,"url":null,"abstract":"<p><p>Current research efforts are underway to create novel approaches for the efficient diagnosis, monitoring, and mitigation of Kyasanur Forest Disease Virus (KFDV) infections. Flavivirus subunit-based vaccines based on envelope glycoprotein EDIII are now in preclinical and clinical research stages. Efficient purification and isolation methods for surface immunogenic viral antigens, including the recombinant envelope immunoglobulin-like domain III (rEDIII) protein, are crucial for the production and manufacturing of promising vaccine candidates that have been extensively assessed in previous literature. Here, we describe a method for high-yield expression, purification, and refolding of a KFDV rEDIII protein from a bacterial expression system. The KFDV rEDIII protein is extracted from the inclusion bodies in urea denaturing buffer followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The purified, denatured KFDV rEDIII protein was subsequently refolded using a step-wise gradient urea dilution via the dialysis method. The circular dichroism and Fourier transform infrared spectroscopy analysis confirms that the refolded KFDV rEDIII maintains the native secondary conformation majorly containing β-strands. Our study provides valuable insights into the design and expression strategies of rEDIII as a novel subunit vaccine candidate against KFDV.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115089"},"PeriodicalIF":2.2000,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of virological methods","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.jviromet.2024.115089","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Current research efforts are underway to create novel approaches for the efficient diagnosis, monitoring, and mitigation of Kyasanur Forest Disease Virus (KFDV) infections. Flavivirus subunit-based vaccines based on envelope glycoprotein EDIII are now in preclinical and clinical research stages. Efficient purification and isolation methods for surface immunogenic viral antigens, including the recombinant envelope immunoglobulin-like domain III (rEDIII) protein, are crucial for the production and manufacturing of promising vaccine candidates that have been extensively assessed in previous literature. Here, we describe a method for high-yield expression, purification, and refolding of a KFDV rEDIII protein from a bacterial expression system. The KFDV rEDIII protein is extracted from the inclusion bodies in urea denaturing buffer followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The purified, denatured KFDV rEDIII protein was subsequently refolded using a step-wise gradient urea dilution via the dialysis method. The circular dichroism and Fourier transform infrared spectroscopy analysis confirms that the refolded KFDV rEDIII maintains the native secondary conformation majorly containing β-strands. Our study provides valuable insights into the design and expression strategies of rEDIII as a novel subunit vaccine candidate against KFDV.
期刊介绍:
The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery.
The methods may include, but not limited to, the study of:
Viral components and morphology-
Virus isolation, propagation and development of viral vectors-
Viral pathogenesis, oncogenesis, vaccines and antivirals-
Virus replication, host-pathogen interactions and responses-
Virus transmission, prevention, control and treatment-
Viral metagenomics and virome-
Virus ecology, adaption and evolution-
Applied virology such as nanotechnology-
Viral diagnosis with novelty and comprehensive evaluation.
We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.