Stable transfection system for Babesia sp. Xinjiang

Parasites & Vectors

Guiquan Guan¹  Junlong Liu¹  Jianxun Luo¹  Youquan Li¹  Hong Yin¹  Jifei Yang¹  Aihong Liu¹  Xiaoxing Wang¹  Jinming Wang¹ 

[1] State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science;

关键词: Babesia sp. Xinjiang;    Stable transfection;    Genetic manipulation;    Cross-over homologous recombination;   

DOI  :  10.1186/s13071-021-04940-x

来源: DOAJ

【 摘 要 】

Abstract Background Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers. Methods We evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites. Results Transfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months. Conclusions In our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj. Graphical abstract

【 授权许可】

Unknown