严重急性呼吸道综合征冠状病毒2全长cDNA的构建新途径  

A new approach for construction of full-length cDNA of SARS-CoV-2

作　　者：安欢欢 钱莎莎 于代冠 申硕 AN Huan-huan;QIAN Sha-sha;YU Dai-guan;SHEN Shuo(Wuhan Institute of Biological Products,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207 [3]湖北省疫苗技术创新中心,湖北武汉430207

出　　处：《中国生物制品学杂志》2021年第12期1418-1426,共9页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC08421000,2020YFC0860600);湖北省重点研发计划(KTSI1800083A)。

摘　　要：目的构建严重急性呼吸道综合征冠状病毒2(severe acute respiratory syndrome coronavirus-2,SARS-CoV-2)全长cDNA,为SARS-CoV-2基因功能、药物筛选和安全减毒灭活疫苗株的研究提供重要工具,也为大基因组RNA病毒cDNA的构建提供新的方法。方法结合In-Fusion、Gibson Assembly以及Recombineering技术对SARS-CoV-2基因组进行全长cDNA克隆的从头合成。首先将SARS-CoV-2全长基因组分17个片段进行扩增,利用In-Fusion试剂盒将这些小片段连接、克隆,以获得插入序列约5000 bp的6个pUC19重组质粒;然后利用Gibson Assembly试剂盒将30000 bp SARS-CoV-2全长基因组分3次逐步连接至pBR322载体,每次连接10000 bp,得到含30000 bp SARSCoV-2全长cDNA的重组质粒;最后利用Recombineering技术修复逐步连接过程中通过NotⅠ位点引入的两处多余GC碱基。结果构建了SARS-CoV-2 WIV04株的全长cDNA,插入基因组的重组质粒经酶切和测序鉴定正确。为区别于野生型毒株SARS-CoV-2 WIV04,分别在全长cDNA的8791 nt和12655 nt进行了同义突变,引入了两处BglⅠ酶切位点,以此作为遗传标记。结论成功构建了SARS-CoV-2全长cDNA,为SARS-CoV-2基础研究和应用研究提供了技术平台。Objective To construct the full-length cDNA of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)so as to provide an important tool for the studies of gene function,drug screening and the development of a safe and attenuated strain for inactivated vaccine production and a novel method for the construction of cDNA of other large genome RNA viruses.Methods In-Fusion,Gibson Assembly and Recombineering were combined to construct the fulllength cDNA of SARS-CoV-2 de novo.Firstly,17 PCR fragments were amplified covering the full length of SARS-CoV-2 genome.These small fragments were joined into six fragments approximately 5000 bp in size and individually cloned into pUC19 vector using In-Fusion kit.Secondly,using Gibson Assembly kit,each pair of 5000 bp fragment was combined and ligated into pBR322 vector through three steps,10000 bp for each,to obtain a recombinant plasmid harboring the full-length genome approximately 30000 bp.Finally,the extra GC bases at two sites introduced by NotI in this construction process were removed by Recombineering technology in a BAC vector.Results The full-length cDNA of SARS-CoV-2 WIV04 strain was constructed,and the recombinant plasmid containing the full genome was constructed correctly as proved by restriction analysis and sequencing.To distinguish the recombinant virus from the wild strain,two synonymous mutations were introduced at sites of 8791 and 12655 nt respectively,producing two BglⅠrestriction sites as genetic markers.Conclusion The full-length cDNA of SARS-CoV-2 was successfully constructed,which provided a technical platform for the basic and application researches of SARS-CoV-2.

关 键 词：严重急性呼吸道综合征冠状病毒2 全长CDNA In-Fusion克隆 Gibson拼接 重组技术 

分 类 号：Q785[生物学—分子生物学]