Das Manjima, Kumar Vivek, Madhukalya Rishav, Gupta Rohit, Agarwal Vidushi, Choudhary Shweta, Bhutkar Mandar, Tomar Shailly, Kumar Dilip, Kumar Rajesh
Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand, India.
Trivedi School of Biosciences, Ashoka University, Sonipat, Haryana, India.
J Virol Methods. 2025 Apr;333:115089. doi: 10.1016/j.jviromet.2024.115089. Epub 2024 Dec 3.
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.
目前正在开展研究工作，以创建针对基孔肯雅森林病病毒（KFDV）感染的高效诊断、监测和缓解的新方法。基于包膜糖蛋白EDIII的黄病毒亚单位疫苗目前正处于临床前和临床研究阶段。高效纯化和分离表面免疫原性病毒抗原的方法，包括重组包膜免疫球蛋白样结构域III（rEDIII）蛋白，对于生产和制造在先前文献中已得到广泛评估的有前景的候选疫苗至关重要。在此，我们描述了一种从细菌表达系统中高产表达、纯化和重折叠KFDV rEDIII蛋白的方法。KFDV rEDIII蛋白在尿素变性缓冲液中从包涵体中提取，随后通过镍-亚氨基三乙酸（Ni-NTA）亲和层析进行纯化。纯化后的变性KFDV rEDIII蛋白随后通过透析法使用逐步梯度尿素稀释进行重折叠。圆二色性和傅里叶变换红外光谱分析证实，重折叠的KFDV rEDIII主要保持了含有β链的天然二级构象。我们的研究为作为抗KFDV新型亚单位疫苗候选物的rEDIII的设计和表达策略提供了有价值的见解。
