功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示<sup>*</sup>

doi:10.13523/j.cb.2203051

中国生物工程杂志

2022, Vol. 42

Issue (6): 30-38 DOI: 10.13523/j.cb.2203051

技术与方法

功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示^*

于璐¹,胡暄¹,张小鹃^1,²,牛安娜¹,张晓鹏^1,^**(

)

1.军事科学院军事医学研究院生物工程研究所北京 100071
2.安徽大学物质科学与信息技术研究院合肥 230601

Surface Display of Functional RBD of SARS-CoV-2 in Pichia pastoris

YU Lu¹,HU Xuan¹,ZHANG Xiao-juan^1,²,NIU An-na¹,ZHANG Xiao-peng^1,^**(

)

1. Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China
2. Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, China

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摘要：

目的:设计并构建新型冠状病毒(SARS-CoV-2)受体结合结构域(receptor binding domain,RBD)在毕赤酵母表面的展示体系,并对表面展示的RBD进行功能性评价,从而为以RBD为靶点的高通量药物筛选平台奠定基础。方法:将四种锚定分子与新冠病毒RBD融合,电转化至毕赤酵母中;通过细胞免疫荧光分析,筛选能够成功展示RBD的锚定系统;进一步分析其与血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)受体的亲和力,证明展示在细胞表面RBD分子的功能。结果:仅Sed1p锚定分子能够有效呈递RBD至毕赤酵母细胞表面,展示效率约为70%;亲和力分析结果表明,ACE2受体和表面展示RBD的亲和力(K_D = 30.42 nmol/L)与溶液中RBD的亲和力(K_D = 16.00 nmol/L)较为接近。结论:这一体系能够在毕赤酵母表面高效地展示具有生物学功能的RBD,可用于抗新冠病毒RBD药物的高通量筛选和评价。

关键词： 表面展示; 新型冠状病毒; 毕赤酵母; 受体结合结构域; 亲和力

Abstract:

Objective: To establish a high-throughput platform for drug discovery targeting receptor binding domain (RBD) of SARS-CoV-2, a surface display system was designed and constructed to deliver functional RBD to the surface of Pichia pastoris. Methods: Four anchor molecules were fused to RBD, and then were transformed into Pichia pastoris by using electroporation. The surface display efficiency of RBD was measured using flow cytometry, and the affinity of RBD binding to the ACE2 receptor was further determined. Results: RBD-Sed1p system exhibited the highest surface display efficiency of 70%. The binding affinity to ACE2 of RBD displayed on the cellular surface (K_D=30.42 nmol/L) was close to that of RBD in solution (K_D=16.00 nmol/L). Conclusion: A surface display system of RBD was successfully developed in Pichia pastoris, which can be used for high-throughput screening and evaluation of anti-COVID-19 drugs.

Key words: Surface display SARS-CoV-2 Pichia pastoris Receptor binding domain(RBD) Affinity

收稿日期: 2022-03-23 出版日期: 2022-07-07

ZTFLH:

Q789

基金资助: *国家自然科学基金(82173787)

通讯作者: 张晓鹏 E-mail: zxp8565@aliyun.com

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引用本文:

于璐,胡暄,张小鹃,牛安娜,张晓鹏. 功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示^*[J]. 中国生物工程杂志, 2022, 42(6): 30-38.

YU Lu,HU Xuan,ZHANG Xiao-juan,NIU An-na,ZHANG Xiao-peng. Surface Display of Functional RBD of SARS-CoV-2 in Pichia pastoris. China Biotechnology, 2022, 42(6): 30-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203051 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/30

图1 四种RBD表面展示质粒的设计与构建

图2 RBD表面展示质粒SacⅠ线性化结果

图3 RBD表面展示重组菌株的菌落PCR鉴定

图4 荧光显微镜和流式细胞术检测RBD展示重组菌株

图5 GS115/RBD-Sed1p展示及检测

图6 流式细胞术分析GS115/RBD-Sed1p与ACE2的结合活性

图7 RBD和ACE2受体结合亲和力

图8 GS115/RBD-Sed1p中和抗体竞争性结合检测

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