基于CRISPR/Cas9技术的弓形虫rop16_(I/III)缺陷虫株的构建及毒力鉴定  

CRISPR/Cas9-based construction of rop16_(I/III) deficient strain of Toxoplasma gondii and its virulence identification

作　　者：王聪 程维晟 刘芳 张焰[1,2] FaustinaPappoe 罗庆礼 闻慧琴 邓芳[3] 徐元宏[1,2] 沈继龙 

机构地区：[1]安徽病原生物学省级实验室和人兽共患病安徽省重点实验室,合肥230032 [2]安徽医科大学第一附属医院检验科,合肥230022 [3]安徽医科大学附属省立肿瘤医院检验科,合肥230001

出　　处：《中国人兽共患病学报》2017年第1期22-26,31,共6页Chinese Journal of Zoonoses

基　　金：国家重点基础研究发展计划(973计划)项目(No.2010CB530001);国家自然科学基金(No.81471983)~~

摘　　要：目的构建并鉴定弓形虫RH株rop16_(I/III)缺陷虫株。方法利用CRISPR-Cas9技术进行构建基因缺陷虫株。运用E-CRISPR数据库设计gRNA,并使用定点突变试剂盒突变pSAG1::Cas9-U6::sgUPRT质粒上的gRNA,构建pSAG1::Cas9-U6::sgrop16质粒。此外将rop16上游序列、乙胺嘧啶抗性基因、rop16下游序列3个片段连接成donorDNA,克隆于pUC19质粒上,PCR扩增donor DNA片段。pSAG1::Cas9-U6::sgrop16质粒和donor DNA片段电穿孔转染弓形虫,电转后悬液接种于HFF-1细胞中,3μmol/L乙胺嘧啶筛选电转后的虫株。PCR和Western blotting鉴定克隆化筛选虫株。吉姆萨染色分别比较RH株和RHΔrop16株对HFF-1细胞的增殖与入侵。并比较RH株和RHΔrop16株分别感染昆明小鼠后小鼠的生存和死亡率。结果经测序比对,成功构建了pSAG1::Cas9-U6::sgrop16质粒和pUC19-donorDNA质粒。PCR鉴定结果显示,DHFR编码(编码乙胺嘧啶抗性基因)序列成功插入至靶点位置,Western blotting分析结果未见RHΔrop16株有Rop16_(I/III)蛋白表达。吉姆萨染色后计数结果表明,RH株感染的细胞内每个纳虫泡内速殖子的平均数显著高于RHΔrop16虫株。毒力试验结果显示,RH株感染的小鼠在第7d即出现死亡,而rop16_(I/III)缺陷株在第9d出现死亡,但两种弓形虫株感染动物在第10d均全部死亡,两组间无统计学差异。结论利用CRISPR-Cas9技术成功构建了rop16_(I/III)缺陷的弓形虫RH虫株,rop16_(I/III)基因敲除对弓形虫RH株毒力无明显影响。Toxoplasma gondii RH rop161/m deficient strain was constructed based on CRSPR/Cas9 technology. E-CRISP database was used to design gRNA; mutation of gRNA in pSAGI： ： Cas9-U6： ： sgUPRT plasmid was performed by using site- directed mutagenesis to construct pSAG1 ： ： Casg-U6 ： ： sgropl6 plasmid. The pUClg-donorDNA plasmid was constructed and fragments were amplified by PCR. The plasmid pSAG1..Cas9-U6 ：sgropl6t/m and donor DNA fragments were eleetroporated in- to T. gondii RH strain. Follow electroporation, suspension was inoculated into human foreskin fibroblast cells （HFF-1）. The3 μmol/L pyrimethamine used to screen the electroporated parasite and monoclone was detected by PCR and Western blotting. The proliieration and invasion of RHaropl6 strain were observed in HFF-1 cells by Giemsa stai- ning. Twenty KM mice were infected with 200 tachyzoite of wild type or RHarop16 parasite, respectively. The animal sur- vival was recorded. The results showed that pSAGl：Casg- U6..sgrop16 plasmid and pUC19-donor DNA plasmid weresuccessfully constructed and confirmed by DNA sequencing. PCR identification proved that DHFR coding sequence was suc- cessfully 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 Arop16 1/m strain, on day 9. However, all animals infected with both strains died on day 10 post-infection. The Arop16 1/m strain of T. gondii was successfully constructed by the CRISPR-Cas9 technology and no differ- ence was noted between wild type and RH△rop16 strain in their virulence to mice.

关 键 词：刚地弓形虫 rop16(I/III) CRISPR/Cas9 RH株 基因缺陷 

分 类 号：R382.5[医药卫生—医学寄生虫学]