Desmecht Daniel, Boshra Hani
Faculty of Veterinary Medicine, Department of Morphology and Pathology, FARAH Research Center, University of Liège, Liège, Belgium.
Faculty of Veterinary Medicine, Department of Pathology, Fundamental and Applied Research for Animals and Health (FARAH), University of Liège, Liège, Belgium.
Methods Mol Biol. 2025;2893:169-180. doi: 10.1007/978-1-0716-4338-9_13.
The recombinant expression and purification of viral proteins are a key component in the study of the immune response of viruses, as well as the creation of diagnostic techniques for the detection of viruses. For structurally simple proteins, one commonly used technique is the production of recombinant proteins in bacterial expression systems, which enable the large-scale synthesis and purification of recombinant viral proteins. In this technique, the cDNA encoding for a viral protein is cloned into a bacterial expression vector (with an appropriate purification tag), produced in a modified bacterial culture, and optimized for maximum protein production in a minimal amount of time. In this chapter, a protocol for the production of Rift Valley fever virus nucleoprotein is described. This protein has been previously shown to highly antigenic (and thus, is used in diagnostic tests), and has also been shown to be a potent inducer of the T-cell response following Rift Valley fever virus infection. The protocol outlined in this chapter describes the cloning of the cDNA of RVFV nucleoprotein into a bacterial expression vector, which also contains a fusion protein for optimal protein expression and solubilization, as well as a poly-histidine tag for efficient purification This chapter also describes the steps required for bacterial transformation, culture, lysis, purification and dialysis of RVFV nucleoprotein, resulting in a recombinant protein preparation, which can be upscaled to produce milligram quantities of protein product, which in turn can be used for downstream immunological and diagnostic applications.
病毒蛋白的重组表达和纯化是病毒免疫反应研究以及病毒检测诊断技术创建的关键组成部分。对于结构简单的蛋白质，一种常用技术是在细菌表达系统中生产重组蛋白，这能够大规模合成和纯化重组病毒蛋白。在该技术中，编码病毒蛋白的cDNA被克隆到细菌表达载体（带有合适的纯化标签）中，在经过改良的细菌培养物中生产，并针对在最短时间内实现最大量蛋白质生产进行优化。在本章中，描述了裂谷热病毒核蛋白的生产方案。该蛋白先前已被证明具有高度抗原性（因此用于诊断测试），并且在裂谷热病毒感染后也被证明是T细胞反应的有效诱导剂。本章概述的方案描述了将裂谷热病毒核蛋白的cDNA克隆到细菌表达载体中，该载体还包含用于最佳蛋白表达和溶解的融合蛋白以及用于高效纯化的多组氨酸标签。本章还描述了裂谷热病毒核蛋白的细菌转化、培养、裂解、纯化和透析所需的步骤，从而得到重组蛋白制剂，该制剂可扩大规模生产毫克量的蛋白产物，进而可用于下游免疫学和诊断应用。
