新型冠状病毒复制子人工合成和应用研究进展

万里川; 王学军; 王升启

doi:10.12211/2096-8280.2023-029

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新型冠状病毒复制子人工合成和应用研究进展

特约评述 | 更新时间：2025-03-20

- 新型冠状病毒复制子人工合成和应用研究进展
- Artificial synthesis and applications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicons
- 合成生物学 2024年5卷第1期页码：174-190
- 作者机构：
  
  军事医学研究院生物信息中心，北京 100850
- 作者简介：
  
  [ "万里川（1973—），男，博士，工作人员。研究方向为疾病相关基因的人工合成。 E-mail：wlichuan@sina.com" ]
  [ "王学军（1981—），男，副研究员，北京市科技新星。研究方向为病毒感染复制模型与生物技术药物疫苗研究。 E-mail：xjwang@bmi.ac.cn" ]
  [ "王升启（1961—），男，研究员，博士生导师。研究方向为合成生物学、分子诊断与治疗研究。 E-mail：sqwang@bmi.ac.cn" ]
- 基金信息：
  
  国家重点研发计划(2018YFA0902300;2018YFA0900802);国家自然科学基金(81830101)
- DOI：10.12211/2096-8280.2023-029
  中图分类号： R373.1
- 收稿：2023-04-10，
  
  修回：2023-11-26，
  
  纸质出版：2024-02-29
- 稿件说明：
移动端阅览
万里川，王学军，王升启. 新型冠状病毒复制子人工合成和应用研究进展［J］. 合成生物学， 2024， 5（1）： 174-190

WAN Lichuan， WANG Xuejun， WANG Shengqi. Artificial synthesis and applications of severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） replicons［J］. Synthetic Biology Journal， 2024， 5（1）： 174-190
万里川，王学军，王升启. 新型冠状病毒复制子人工合成和应用研究进展［J］. 合成生物学， 2024， 5（1）： 174-190 DOI： 10.12211/2096-8280.2023-029.

WAN Lichuan， WANG Xuejun， WANG Shengqi. Artificial synthesis and applications of severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） replicons［J］. Synthetic Biology Journal， 2024， 5（1）： 174-190 DOI： 10.12211/2096-8280.2023-029.

摘要

从2019年12月初首次报道到2023年9月，新型冠状病毒在全世界已造成约7.7亿人感染和696万人死亡。由于该病毒具有极强的传染性，理论上相关的研究工作都必须在生物安全三级或以上的实验室中进行。为了克服这一局限性，研究人员应用反向遗传学技术构建了众多复制子，使得在生物安全二级实验室中就可以进行相关的研究工作。科学家们通常会在复制子中插入绿色荧光蛋白或荧光素酶等报告基因以及遗传霉素等抗性基因，方便检测病毒和建立稳定的细胞株。另外，新冠病毒复制子还可通过与共转染的病毒糖蛋白的反式互补作用形成只能单次侵染的病毒粒子，使之能侵染无新冠病毒受体的细胞。因此，本文总结了合成新冠病毒复制子的主要方法，如通过Ⅱ型或ⅡS型限制性内切酶进行体外连接、基于细菌人工染色体的构建、利用酵母转化相关重组进行克隆和应用环形聚合酶延伸反应构建，同时综述了新冠病毒的单周期和稳定表达复制子系统。总之，多种复制子系统的构建和应用为研究新冠病毒基因功能、病毒与宿主互作机制和抗病毒药物的高通量筛选奠定了坚实的基础，为阻止病毒的蔓延和保卫人类的健康做出了重大贡献。

Abstract

From its first report in late 2019 to September 2023

the Corona Virus Disease 2019 (COVID-19) pandemic had accumulatively infected at least 770 million people worldwide

and caused a death toll surpassing 6.96 million. The super-spreading of COVID-19 posed a huge challenge to healthcare systems all over the world

and led to a global economic recession. Later research confirmed that the causative agent is a novel coronavirus

nomenclature Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This virus possesses a similar genomic structure

and displays many common harmful characteristics of SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Due to its acute contagiousness

relevant genetic research and antiviral drug development must be carried out in laboratories with the biosafety level 3 or above

which is insufficient worldwide. Moreover

cost for such experiments is very high

and the processes are tedious

time-consuming and laborious. To overc

ome these limitations and enable relevant research work conducted in laboratories with the biosafety level 2

global researchers have constructed a series of SARS-CoV-2 replicons using cutting-edge reverse genetic techniques. By

in vitro

transcription and electroporation or liposome-related transfection

mRNAs or plasmids carrying replicons can be delivered into diverse susceptible host cells. These replicons can replicate

transcribe

and translate inside permissive cells through their transcription and translation machines. With one or more structural genes deleted

no functional virion can form to lose virus infectivity. At the same time

reporter genes

such as green fluorescent protein (GFP) and/or luciferase gene

can be inserted into the replicons to report the levels of virus replication and transcription

indicating the functions of genes or the effectiveness and efficacy of antiviral compounds. By co-transfection with trans-complemental proteins

such as stomatitis virus glycoprotein

virions can be formed with a feature of the single infection of cells without SARS-CoV-2 receptors

such as angiotensin-converting enzyme 2 (ACE2)

or transmembrane serine protease 2 (TMPRSS2). Resistance-selecting genes can be introduced into the proper position of replicons to select stable cell lines expressing replicons. Thus

cell lines stably expressing replicons can be obtained through multiple passages in the presence of selectable drugs

such as G418. In this article

we summarize synthesis methods for constructing SARS-CoV-2 replicons

such as in vitro ligation by type Ⅱ or ⅡS restriction enzymes

Bacterial Artificial Chromosome (BAC)

yeast transformation-associated recombination (TAR)

and cyclic polymerase extension reactions (CPER). Moreover

we review single-cycle and stably expressed SARS-CoV-2 replicon systems

which provide a solid foundation for studying the SARS-CoV-2 gene function

pathogenesis

virus-host interactions

vaccine assessment

and large-scale high-throughput screening of a

ntiviral drugs to contain transmission of this pandemic.

关键词

Keywords

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