牛干扰素-γ高效无细胞表达系统建立及其功能活性表征

Establishment and functional characterization of an efficient cell-free expression system of bovine interferon-gamma

摘要:

本研究旨在优化pET-23b质粒结构以增加无细胞系统表达牛干扰素-γ (interferon-gamma, IFN-γ)的产量。首先利用生物信息学的方法对牛IFN-γ基因进行稳定性突变，对携带优化后的牛IFN-γ基因的原始质粒origin-pET-23b-IFN-γ通过重叠PCR删除质粒中的rop片段、bom片段和f1 ori片段，构建了高拷贝数m-pET-23b-IFN-γ质粒。用m-pET-23b-IFN-γ质粒进行大肠杆菌无细胞体系表达。经Western blotting分析，表达的牛IFN-γ蛋白具有良好的反应性，并且表达量高于未改造的pET-23b无细胞表达系统。将无细胞表达体系反应的产物离心后取上清，使用His标签蛋白纯化琼脂糖磁珠试剂盒对上清中的His标签蛋白进行纯化，对纯化后的重组牛IFN-γ (rIFN-γ)进行细胞安全性评估、抗牛分枝杆菌的生物活性测定及细胞干扰素刺激基因(interferon-stimulated genes, ISGs)转录水平的定量实时聚合酶链式反应(quantitative real-time PCR, qPCR)测定。结果表明牛IFN-γ在浓度低于100 μg/mL时对细胞几乎无毒性，并且具有抗牛分枝杆菌的活性，ISGs转录水平均升高，说明重组牛IFN-γ具有良好的生物学活性。本研究成功建立了高效无细胞牛IFN-γ表达系统，为牛结核病的诊断与治疗提供了高效、安全的牛IFN-γ生产途径，展现出了广阔的应用前景。

Abstract:

This study aimed to optimize the pET-23b plasmid structure to enhance the bovine interferon-gamma (IFN-γ) yield in a cell-free expression system. Initially, the gene stability of bovine IFN-γ was improved via bioinformatic mutation, The rop fragment, bom fragment and f1 ori fragment in the original plasmid origin-pET-23b-IFN-γ carrying the optimized bovine IFN-γ gene were deleted by overlapping PCR to construct a high copy number m-pET-23b-IFN-γ plasmid. The m-pET-23b-IFN-γ plasmid was subsequently expressed in Escherichia coli-based cell-free expression system. Western blotting confirmed satisfactory reactivity of the expressed bovine IFN-γ, with expression levels exceeding those from the original pET-23b system. Following centrifugation, the supernatant from the cell-free expression system was collected, using magnetic agarose beads for His-tag protein purification kit to purify the His-tagged protein in the supernatant. The purified recombinant bovine IFN-γ (rIFN-γ) was then evaluated for cell safety, biological activity against Mycobacterium bovis, and transcription levels of interferon-stimulated genes (ISGs) in cells. The results showed that bovine IFN-γ was almost non-toxic to cells at the concentration below 100 μg/mL, had anti-M. bovis activity, and increased the transcription levels of ISGs, which indicated that rIFN-γ had good biological activity. We successfully established an efficient cell-free expression system for bovine IFN-γ and offered a safe and productive pathway for bovine IFN-γ generation, relevant for the diagnosis and treatment of bovine tuberculosis, thus indicating broad application prospects.