Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy

Frontiers in Cellular and Infection Microbiology

Junlong Zhao¹  Lan He¹  Sen Wang²  Dongfang Li²  Fangwei Chen²  Wanxin Luo²  Weijun Jiang²  Guan Zhu³ 

[1] Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, China;Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China;Key Laboratory of Zoonosis Research of the Ministry of Education, the Institute of Zoonosis, and the College of Veterinary Medicine, Jilin University, Changchun, China;State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China;

关键词: Babesia duncani;    stable transfection;    gene manipulation;    homologous recombination;    TPX-1;   

DOI  :  10.3389/fcimb.2022.844498

来源: DOAJ

【 摘 要 】

Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of Babesia duncani, a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene elongation factor-1α (ef-1α) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the ef-1αB promoter and the reporter gene mCherry and achieved stable transfection through homologous recombination to integrate the selection marker hDHFR-eGFP into the parasite genome. The potential of this genetic modification method was tested by knocking out the thioredoxin peroxidase-1 (TPX-1) gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the TPX-1 knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for B. duncani, which may facilitate gene function research and vaccine development of B. duncani.

【 授权许可】

Unknown