喀山努尔森林疫病病毒主要包膜糖蛋白EDIII结构域的纯化与鉴定。

IF 2.2 4区 医学 Q3 BIOCHEMICAL RESEARCH METHODS
Manjima Das , Vivek Kumar , Rishav Madhukalya , Rohit Gupta , Vidushi Agarwal , Shweta Choudhary , Mandar Bhutkar , Shailly Tomar , Dilip Kumar , Rajesh Kumar
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引用次数: 0

摘要

目前正在进行的研究工作是为有效诊断、监测和减轻Kyasanur森林疫病病毒(KFDV)感染创造新方法。基于包膜糖蛋白EDIII的黄病毒亚单位疫苗目前处于临床前和临床研究阶段。表面免疫原性病毒抗原(包括重组包膜免疫球蛋白样结构域III (rEDIII)蛋白)的高效纯化和分离方法对于生产和制造有希望的候选疫苗至关重要,这些候选疫苗已在先前的文献中得到广泛评估。在这里,我们描述了一种从细菌表达系统中高效表达、纯化和重折叠KFDV rEDIII蛋白的方法。在尿素变性缓冲液中从包涵体中提取KFDV rEDIII蛋白,并采用镍-硝基三乙酸亲和层析。纯化的,变性的KFDV rEDIII蛋白随后通过透析方法使用逐步梯度尿素稀释重新折叠。圆二色性和傅里叶变换红外光谱分析证实,重新折叠的KFDV rEDIII保持了主要含有β-链的天然二级构象。我们的研究为rEDIII作为一种新的抗KFDV亚单位候选疫苗的设计和表达策略提供了有价值的见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Purification and characterization of kyasanur forest disease virus EDIII domain of major envelope glycoprotein
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.
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来源期刊
CiteScore
5.80
自引率
0.00%
发文量
209
审稿时长
41 days
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.
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