背景:SARS-CoV2病毒,负责COVID-19大流行,有4种结构蛋白和16种非结构蛋白。S蛋白是暴露在病毒表面的结构蛋白之一,是产生中和抗体和疫苗的主要靶标。S蛋白形成三聚体,可通过其受体结合域(RBD)结合血管紧张素转化酶2(ACE2)以进入细胞。
目的:本研究的目的是在HEK293细胞中以组成型和稳定的方式表达一种新的RBD重组蛋白,以便将其用作COVID-19的替代免疫原和诊断工具。
方法:该蛋白被设计为含有免疫球蛋白信号序列,RBD的外植体C端部分,负责噬菌体T4三聚诱导物的区域,和pCDNA-3.1质粒中的六个组氨酸。改造后,用遗传霉素-G418筛选细胞,并使用Ni2+-agarand大小排阻层析从血清培养上清液中纯化.通过交联和圆二色性实验对蛋白质进行了结构鉴定,并用AS03或明矾佐剂免疫小鼠。检查小鼠血清的抗体识别,受体结合抑制,和病毒中和,而在存在RBD的情况下评估脾脏的γ-干扰素产生。
结果:细胞培养上清液中释放的蛋白质,并且表现出135kDa的分子量,具有类似于单体和三聚体RBD的二级结构。纯化后,它形成了包含三聚体和六聚体的多聚体结构,能够结合ACE2受体。当与AS03佐剂组合时,其在小鼠中产生高抗体滴度(高达1:50,000)。血清能够抑制生物素标记的ACE2与病毒S1亚基的结合,并且可以以高达1:2000的稀释率中和武汉病毒株进入细胞。它在免疫小鼠脾细胞中产生特异性IFN-γ产生细胞。
结论:我们的数据描述了一种新的含有RBD的蛋白质,形成三聚体和六聚体,能够诱导针对SARS-CoV2的保护性体液和细胞反应。
结论:这些结果为对抗COVID-19增加了新的武器库,作为诊断的替代免疫原或抗原。
BACKGROUND: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry.
OBJECTIVE: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19.
METHODS: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD.
RESULTS: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes.
CONCLUSIONS: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2.
CONCLUSIONS: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.