Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus.
Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus.
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DOI：
10.1002/9780471729259.mc15e01s21
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发表时间：
2011-05
影响因子：
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通讯作者：
Liu, Pinghua
中科院分区：
文献类型：
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作者：
Leibowitz, Julian;Kaufman, Gili;Liu, Pinghua
该方案的重点是小鼠肝炎病毒(MHV)，偶尔会提到其他冠状病毒。这些协议中的许多都可以很容易地适应其他冠状病毒。将介绍在DBT和17CL-1细胞中繁殖MHV的方案；病毒库的储存和滴定；蔗糖梯度纯化MHV；以及通过cDNA组装方法和靶向重组产生重组病毒。还包括用于生长和滴定MHV的DBT、17CL-1和L2细胞的增殖，以及BHK-R细胞和FCWF细胞的生长。后两种细胞系分别通过体外基因组装方案和靶向重组方案用于再生感染性MHV，从而允许对这些病毒进行反向遗传操作。还将提出一项用于维护用于产生重组MHV的大质粒的附加方案。货币。普罗托克。微生物。21：15欧洲标准时间1.1-15欧洲标准时间1.46©2011 John Wiley&Sons，Inc.
The focus of this protocol is mouse hepatitis virus (MHV), with occasional references to other coronaviruses. Many of these protocols can be easily adapted to other coronaviruses. Protocols for propagating MHV in DBT and 17CL‐1 cells; the storage and titration of viral stocks; purification of MHV on sucrose gradients; and the generation of recombinant viruses by a cDNA assembly method and by targeted recombination will be presented. Protocols are also included for the propagation of DBT, 17CL‐1, and L2 cells used for growing and titrating MHV, and for the growth of BHK‐R cells and FCWF cells. The latter two cell lines are used for regenerating infectious MHV by an in vitro cDNA assembly protocol and by a targeted recombination protocol, respectively, allowing reverse genetic manipulation of these viruses. An additional protocol for the maintenance of the large plasmids used for generating recombinant MHVs will also be presented. Curr. Protoc. Microbiol. 21:15E.1.1‐15E.1.46. © 2011 by John Wiley & Sons, Inc.