背景：大肠杆菌产生的重组过敏原（E.大肠杆菌）系统在过敏和疫苗开发的成分解析诊断中起着重要作用。然而,重组过敏原的不正确折叠可能会影响其应用。因此,监测重组过敏原的正确折叠非常重要。目前,目前仍缺乏解决这一问题的质量控制策略。在这项研究中,螨过敏原,以Derf2为例，建立了一种新的质量控制策略，基于色谱法分离过敏原，并在酶联免疫吸附试验上验证分离的过敏原的IgE反应性。
方法：对编码Derf2的核苷酸序列进行密码子优化，克隆到pET-28a(+)质粒中。寻找在大肠杆菌中表达Derf2的最佳条件。Derf2的包涵体变性并用镍亲和色谱法纯化。使用谷胱甘肽氧化还原系统比较了重折叠过程。通过阴离子交换色谱分离完全和部分折叠的蛋白质，并通过间接酶联免疫吸附试验验证了分离蛋白的IgE反应性。
结果：在大肠杆菌中成功表达了Derf2编码基因的优化的387bp片段。最佳诱导条件包括预诱导细菌密度，600nm处的吸光度值为0.6，1mM异丙基β-d-硫代半乳糖苷在28℃下4h。重折叠后的Derf2蛋白通过色谱法分离，获得两个级分。通过尺寸排阻色谱法将第一部分鉴定为单体蛋白质，将第二部分鉴定为聚集体。间接酶联免疫吸附测定也证实第一部分显示较高的IgE反应性。
结论：在这项研究中，以螨Derf2为例，建立了基于色谱分离和IgE反应性监测的新型质量控制策略，首次系统评价了多种制备方法的有效性。与现有方法如尺寸排阻色谱法相比，它更快、更方便。该策略为大肠杆菌产生的重组过敏原在成分分辨诊断中的稳定应用以及未来分子疫苗的开发奠定了基础。
BACKGROUND: Recombinant allergens produced by Escherichia coli (E. coli) system play an important role in the component-resolved diagnostics of allergy and vaccine development. However, incorrect folding of recombinant allergens may affect their application. Therefore, it is very important to monitor the correct folding of recombinant allergens. Currently, there is still a lack of a quality control strategy to solve this problem. In this study, a mite allergen, Der f 2, was taken as an example to establish a novel quality control strategy, which was based on chromatography to isolate the allergen, and on enzyme-linked immunosorbent assay to verify the IgE reactivity of the isolated allergen.
METHODS: The nucleotide sequence encoding Der f 2 was codon-optimized and cloned into pET-28a (+) plasmid. Best conditions for the expression of Der f 2 in E. coli were sought. The inclusion body of Der f 2 was denatured and purified by nickel affinity chromatography. Refolding processes were compared using glutathione redox system. The fully and partially folded proteins were separated by anion exchange chromatography, and the IgE reactivity of the isolated proteins was verified by indirect enzyme-linked immunosorbent assay.
RESULTS: An optimized 387 bp segment of the Der f 2 coding gene was successfully expressed in E. coli. Best induction conditions included preinduction bacterial density with absorbance value at 600 nm was 0.6, 1 mM isopropyl beta-
d-thiogalactopyranoside at 28°C for 4 h. The Der f 2 protein after refolding was separated by chromatography and two fractions were obtained. The first fraction was identified as monomer protein and the second as aggregate by size-exclusion chromatography. Indirect enzyme-linked immunosorbent assay also confirmed that the first fraction showed higher IgE reactivity.
CONCLUSIONS: In this study, a novel quality control strategy based on chromatographic separation and IgE reactivity monitoring was established in the
case of mite Der f 2, which systematically evaluated the effectiveness of multiple preparation methods for the first time. It is faster and more convenient when compared with the existing methods such as size-exclusion chromatography. This strategy laid a foundation for the stable application of recombinant allergens produced by E. coli in component-resolved diagnostics and the development of molecular vaccines in the future.
