摘要
瞄准：日冕病毒的尖铁蛋白质为病毒绑定，熔化和入口负责，并且是中和抗体的主要 inducer。这篇论文是发现严重尖锐呼吸联系症候群的日冕病毒(SARS-Cov ) 的一个可溶、功能的 recombinant 受体绑定领域，并且分析它的受体绑定能力。方法：三个熔化标签(谷胱甘肽 S-transferase， GST；thioredoxin， Trx；麦芽糖绑定蛋白质， MBP ) ，它最好贡献增加的溶解度并且到便于异种蛋白质的合适的合拢，被用来在埃希氏杆菌属关口 i 获得 RBD 蛋白质的可溶、功能的表示(BL21 (DE3 ) 和 Rosetta-gamiB (DE3 ) 拉紧) 。净化的可溶的 RBD 蛋白质的受体绑定能力然后被 ELISA 和流动血细胞计数试金检测。结果：当在许多不同文化和正式就职条件下面在 BL21 (DE3 ) 和 Rosetta-gamiB (DE3 ) 作为 TrxA 标签形式熔化了时， SARS-Cov 尖铁蛋白质的 RBD 被表示为包括身体。并且当 RBD 被表示时，当 MBP 标注了形式， SDS 页上没有可见表示乐队。仅仅 GST 标注了 RBD 是可溶的在 BL21 (DE3 ) 表示了，并且蛋白质被巢菜净化主要层析系统。ELISA 数据证明那 GST/RBD 抗原带着 anti-RBD 老鼠有阳性反应单音的同种细胞的抗体 1A5。进一步的流动血细胞计数试金表明了 RBD ACE2 的有约束力的能力的高效率(变换血管收缩素的酶 2 ) 积极 Vero E6 房间。并且 ACE2 与 SARS-Cov 尖铁蛋白质作为反省的细胞的受体被证明一个起始亲密关系的相互作用。到 Vero E6 房间的 GST 和 GST/RBD 绑定的几何平均数分别地是 77.08 和 352.73。结论：在这篇论文，我们得到标注 RBD 蛋白质在 E.coli BL21 (DE3 ) 表示了的足够的可溶的 N 终端 GST；从 ELISA 和流动血细胞计数试金的数据证明 recombinant 蛋白质高效地对 ACE2 积极 Vero E6 房间功能、有约束力。并且从 E.coli 导出的 recombinant RBD 能习惯于与受体开发对块 S 蛋白质绑定疫苗的子单元并且到抵销 SARS-Cov 感染。
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 AKTA 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 EcoliBL21 （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.
