用植物生产含有两种 SARS-CoV-2 变体受体结合域 (RBD) 的重组抗原

IF 2 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Flavia Fagiani, Rachele Frigerio, Anna Maria Salzano, Andrea Scaloni, Carla Marusic, Marcello Donini
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引用次数: 0

摘要

目的 这项工作的目的是在plats中快速生产两种重组抗原(RBDw-Fc和RBDo-Fc),这两种抗原含有SARS-CoV-2变体武汉和Omicron的尖峰(S)蛋白的受体结合域(RBD),作为与小鼠IgG2a抗体恒定区(Fc)的Fc部分的融合蛋白。结果这两种重组抗原通过真空农业过滤在烟草植物中表达,并显示出相当的纯化率(约 35 毫克/千克叶片鲜重)。结论 它们的 Western 印迹和 Coomassie 染色证明,在 RBD 和 Fc 之间的区域发生了主要的植物体内蛋白水解,这在 RBDw-Fc 中尤为明显,RBDw-Fc 是唯一带有 HRV 3C 半胱氨酸蛋白酶识别位点的抗原。两个 RBD N-连接糖基化位点显示出非常均匀的轮廓,不含植物典型糖,最丰富的糖基化形式是复合糖 GlcNAc4Man3。在 Western 印迹分析中,抗 SARS-CoV-2 人类中和单克隆抗体 J08-MUT 能特异性识别这两种抗原,RBDw-Fc 还被成功用于与血管紧张素转换酶 2 受体结合的竞争性 ELISA 实验,以验证疫苗接种患者血清的中和能力。这两种与鼠 Fc 区融合的 SARS-Cov-2 抗原都是在植物中快速生产出来的,而且功能正常,在诊断中具有潜在的应用价值。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants

Objectives

The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc).

Results

The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35 mg/kg leaf fresh weight).

Conclusions

Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.

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来源期刊
Biotechnology Letters
Biotechnology Letters 工程技术-生物工程与应用微生物
CiteScore
5.90
自引率
3.70%
发文量
108
审稿时长
1.2 months
期刊介绍: Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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