非复制型弓形虫的构建及其对小鼠致病性分析

Construction of nonreplicating Toxoplasma and its pathogenicity in mice

关键词:  非复制型尿嘧啶营养缺陷型弓形虫  CRISPR-Cas9  小鼠  致病性

Key words:  nonreplicating Toxoplasma uracil auxotrophs  CRISPR-Cas9 technology  mice  pathogenicity

摘要:

为研究非复制型弓形虫对小鼠的致病性，利用CRISPR-Cas9技术构建非复制型尿嘧啶营养缺陷型弓形虫(NRTUAs)，通过药物和荧光筛选阳性虫株，PCR鉴定，并以噬斑试验鉴定NRTUAs株的表型；将NRTUAs株急性感染小鼠，并以△KU80株处理为感染对照，PBS处理为空白对照，每日记录小鼠的死亡情况，并对各组小鼠器官进行病理组织学观察与分析。结果表明:1)NRTUAs株对乙胺嘧啶和氯霉素耐药，重组同源臂上下游PCR鉴定均为阳性，内源性基因位点缺失PCR鉴定均为阴性；2)NRTUAs株在含有尿嘧啶的培养基中能够生长且产生噬斑，在缺乏尿嘧啶的培养基中不能生长且不产生噬斑；3)NRTUAs感染小鼠后，全部存活，且PBS组小鼠的器官组织学形态一致；△KU80组小鼠全部死亡，且两组小鼠器官出现严重病理变化。综上，本研究成功构建非复制型NRTUAs株，该虫株对小鼠无致死性，且小鼠各器官无病理变化，为NRTUAs作为弓形虫疫苗候选株在家畜养殖中的应用奠定理论基础。

Abstract:

In order to explore the pathogenicity of nonreplicating Toxoplasma in mice, nonreplicating Toxoplasma uracil auxotrophs(NRTUAs)were constructed by CRISPR-Cas9. The positive strains were selected by drugs and fluorescence, and identified by PCR. The phenotype of NRTUAs was identified by plaque assay. Mice were acutely infected with NRTUAs. Mice treated with△KU80 strains were used as infection control, while mice treated with PBS were utilized as blank control. The death of mice was recorded daily and the organs were analyzed by histopathology. The results indicated that: 1)NRTUAs were resistant to pyrimidine and chloramphenicol, and the PCR identification of upstream and downstream of the recombinant homologous arm was positive, while the PCR identification of endogenous gene deletion was negative; 2)NRTUAs could grow and produce plaques in the medium containing uracil, but could not grow and produce plaques in the medium lacking uracil; 3)After NRTUAs infection, all the mice survived, and the histological morphology was the same as the PBS group. All the mice in △KU80 group died, and the organs showed serious pathological changes. In summary, NRTUAs strains were successfully constructed in this study, which was not fatal to mice and had no pathological changes in various organs of mice. This study laid a theoretical foundation for the application of NRTUAs as candidate strains of Toxoplasma gondii vaccine in livestock breeding.