弓形虫子孢子抗原SporoSAG基因缺失虫株的构建

Construction of Toxoplasma gondii Strain Lacking of SporoSAG Antigen Gene

随着近几年基因编辑技术的不断更新迭代,特别是CRISPR/Cas9系统的出现,基因编辑的效率和特异性得到了极大的提高。本研究旨在运用新一代基因编辑技术CRISPR/Cas9对子孢子时期特异表达的编码子孢子表面抗原的Sporo SAG基因进行敲除。将相应的DNA元件转到虫体细胞后,通过药物筛选、单克隆的挑选和鉴定,结果表明成功获得sporosag敲除虫株。同时也证明,该CRISPR/Cas9基因编辑系统可以应用于弓形虫子孢子形成相关基因的敲除和改造。

In recent years,with the evolution and improvement of genome editing technologies,especially the emergence of CRISPR/Cas9 system,the efficiency and specificity of gene editing have been greatly improved.This study aimed to knock out the SporoSAG gene encoding a toxoplasma sporozoite specific surface antigen,using a new generation of gene editing technology CRISPR/Cas9.After introducing the corresponding DNA elements into typeⅠstrain RH by electroporation,transfectants were drug screened and single cloned by limiting dilution.Subsequently knockout mutants were identified by diagnostic PCR.The resuLts showed that the SporoSAG gene knockout strain was successfuLly constructed.This study also proved that the CRISPR/Cas9 gene editing system couLd be applied to edit genes related to sporozoite development of Toxoplasma gondii.