中国人兽共患病学报2017，Vol.33Issue(1)：22-26,31,6.DOI:10.3969/j.issn.1002-2694.2017.01.004
基于CRISPRR/Cas9技术的弓形虫rop16Ⅰ/Ⅲ缺陷虫株的构建及毒力鉴定
CRISPR/Cas9-based construction of rop16Ⅰ/Ⅲ deficient strain of Toxoplasma gondii and its virulence identification
摘要
Abstract
Toxoplasma gondii RH rop16Ⅰ/Ⅲ deficient strain was constructed based on CRSPR/Cas9 technology.E-CRISP database was used to design gRNA;mutation of gRNA in pSAG1:∷Cas9-U6∷ sgUPRT plasmid was performed by using sitedirected mutagenesis to construct pSAG1∷Cas9-U6∷sgrop16 plasmid.The pUC19-donorDNA plasmid was constructed and fragments were amplified by PCR.The plasmid pSAG1:Cas9-U6:sgrop16Ⅰ/Ⅲ and donor DNA fragments were electroporated into T.gondii RH strain.Follow electroporation,suspension was inoculated into human foreskin fibroblast cells (HFF-1).The 3 μmol/L pyrimethamine was used to screen the electroporated parasite and monoclone was detected by PCR and Western blotting.The proliferation and invasion of RH△ropo16 strain were observed in HFF-1 cells by Giemsa staining.Twenty KM mice were infected with 200 tachyzoite of wild type or RH△rop16 parasite,respectively.The animal survival was recorded.The results showed that pSAG1:Cas9-U6:sgrop16 plasmid and pUC19-donor DNA plasmid were successfully constructed and confirmed by DNA sequencing.PCR identification proved that DHFR coding scquence was successfully inserted to the target position.Western blotting analysis revealed deficient expression of ROP16 in the RH△rop16 strain.Giemsa staining indicated that the number of parasites per parasite phorous vacuole of wild type Toxoplasma-infected cells was more than that of the RH△rop16 infected cells.Virulence examination indicated that,the wild type strain infected mice began to die on day 7 post-infection where as Arop16Ⅰ/Ⅲ strain,on day 9.However,all animals infected with both strains died on day 10 post-infection.The Arop16Ⅰ/Ⅲ strain of T.gondii was successfully constructed by the CRISPR-Cas9 technology and no difference was noted between wild type and RHArop16 strain in their virulence to mice.关键词
刚地弓形虫/rop16Ⅰ/Ⅲ/CRISPR/Cas9/RH株/基因缺陷Key words
Toxoplasma gondii/rop16Ⅰ/Ⅲ/CRISPR/Cas9/RH strain/gene knockout分类
医药卫生引用本文复制引用
王聪,沈继龙,程维晟,刘芳,张焰,Faustina Pappoe,罗庆礼,闻慧琴,邓芳,徐元宏..基于CRISPRR/Cas9技术的弓形虫rop16Ⅰ/Ⅲ缺陷虫株的构建及毒力鉴定[J].中国人兽共患病学报,2017,33(1):22-26,31,6.基金项目
Supported by the National Natural Science Foundation of China (No.81471983) and the National Basic Science Research Program of China (No.2010CB530001)国家重点基础研究发展计划(973计划)项目(No.2010CB530001) （No.81471983）
国家自然科学基金(No.81471983) （No.81471983）
