高效重组痘苗病毒天坛株载体系统的建立  

Establishment of an Efficient Recombinant Vaccinia Virus Tiantan Strain Vector System

作　　者：张迪 许丰雯 熊梓辰 郭斐[1] ZHANG Di;XU Fengwen;XIONG Ziehen;GUO Fei(NHC,Key Laboratory of Systems Biology of Pathogens,Institute of Pathogen Biology,Chinese Aacdemy of Medical Science,Peking Union Medical College,Beijing 100176,China)

机构地区：[1]中国医学科学院北京协和医学院病原生物学研究所国家卫生健康委员会病原系统生物学重点实验室,北京100176

出　　处：《病毒学报》2019年第2期175-182,共8页Chinese Journal of Virology

摘　　要：以痘苗病毒为载体的溶瘤病毒作为癌症治疗的新方向效果显著。同时,痘苗病毒还可以作为疫苗载体抵抗HIV、H5N1等病毒感染。因技术限制,重组痘苗病毒主要通过同源重组的方法获得,但此方法获得重组病毒的效率较低。随着CRISPR(Clustered regularly interspaced short palindromic repeat)-Cas9技术成功应用于多种动物、组织和细胞的基因编辑中,本研究拟利用CRISPR-Cas9技术建立高效重组痘苗载体系统。本研究首先构建了三个靶向痘苗病毒天坛株TK区的gRNA-Cas9的质粒以及重组质粒pJ2R-EGFP,通过瞬时转染gRNA-Cas9或建立gRNA-Cas9稳定细胞系后,导入重组质粒pJ2R-EGFP并感染VTT,获得表达EGFP的重组病毒。此高效重组痘苗病毒载体系统的重组效率比传统的同源重组方法大大提高。因此,本研究建立的高效痘苗病毒载体重组系统,为其在疫苗载体构建、肿瘤免疫治疗等方面提供参考价值。Vaccinia virus can be used as a carrier for oncolytic virus as a new direction of cancer treatment, which is of great effective.At the same time, vaccinia virus has also been used as a vaccine vector against diseases such as HIV-1, H5N1.Due to technical limitations, recombinant virus was obtained mainly by homologous recombination, the efficiency of which was extremely low.With the successful application of CRISPR( clustered regularly interspaced short palindromic repeat)-Cas9 in a variety of animals, tissues and cells, this study intended to use the CRISPR-Cas9 to establish an efficient recombinant vaccinia vector system.Using three gRNA-Cas9 plasmids targeting TK region of the Tiantan strain, the recombinant viruses expressing EGFP were obtained by introducing gRNA-Cas9 plasmids, or using gRNA-Cas9 stable cell lines, together with repair donor pJ2R-EGFP and VTT infection.The recombination efficiency was much higher than traditional homologous recombination.This system greatly improves the efficiency of recombination of vaccinia virus vector, and provides reference value for vaccine vector con structio n and tumor immuno therapy.

关 键 词：痘苗病毒 CRISPR-Cas9 高效重组系统 溶瘤病毒 疫苗载体 

分 类 号：R373.9[医药卫生—病原生物学] R511[医药卫生—基础医学]