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首页> 外文期刊>World Journal of Gastroenterology >Receptor-bind ing domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization
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Receptor-bind ing domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

机译：SARS-Cov穗突蛋白的受体结合域：在大肠杆菌中的可溶性表达，纯化和功能表征

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AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli(BL21(DE3) and Rosetta-gamiB (DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AEKTA Prime Chromatography system. The ELISA data showed that GST·RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST·RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21 (DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.
机译：目的：寻找重症急性呼吸综合征相关冠状病毒（SARS-Cov）的可溶性和功能性重组受体结合域，并分析其受体结合能力。方法：使用三种融合标签（谷胱甘肽S-转移酶，GST，硫氧还蛋白，Trx，麦芽糖结合蛋白，MBP）来获得可溶性和功能性表达，这些融合标签优选有助于增加溶解度并促进杂蛋白的正确折叠。大肠杆菌中的RBD蛋白（BL21（DE3）和Rosetta-gamiB（DE3）菌株）。然后通过ELISA和流式细胞术测定纯化的可溶性RBD蛋白的受体结合能力。结果：在许多不同的培养和诱导条件下，当以TrxA标签形式融合在BL21（DE3）和Rosetta-gamiB（DE3）中时，SARS-Cov刺突蛋白的RBD表达为包涵体。当RBD以MBP标记形式表达时，在SDS-PAGE上没有可见的表达带。只有GST标记的RBD可溶于BL21（DE3）中表达，并且该蛋白质通过AEKTA Prime色谱系统纯化。 ELISA数据显示GST·RBD抗原与抗RBD小鼠单克隆抗体1A5呈阳性反应。进一步的流式细胞术分析证明RBD与ACE2（血管紧张素转换酶2）阳性Vero E6细胞的结合能力非常有效。 ACE2被证明是一种细胞受体，可冥想与SARS-Cov穗突蛋白的初始亲和力相互作用。 GST和GST·RBD与Vero E6细胞结合的几何平均值分别为77.08和352.73。结论：本文获得了在大肠杆菌BL21（DE3）中表达的足够的可溶性N末端GST标记的RBD蛋白。 ELISA和流式细胞仪检测得到的数据表明，重组蛋白具有功能并与ACE2阳性Vero E6细胞有效结合。来源于大肠杆菌的重组RBD可用于开发亚单位疫苗，以阻断S蛋白与受体的结合并中和SARS-Cov感染。

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