Stable transfection system for Babesia sp. Xinjiang

文献类型： 外文期刊

第一作者： Wang, Jinming

作者： Wang, Jinming;Wang, Xiaoxing;Guan, Guiquan;Yang, Jifei;Liu, Junlong;Liu, Aihong;Li, Youquan;Luo, Jianxun;Yin, Hong;Yin, Hong

作者机构：

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

期刊名称：PARASITES & VECTORS （ 影响因子：3.876； 五年影响因子：3.959 ）

ISSN： 1756-3305

年卷期： 2021 年 14 卷 1 期

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收录情况： SCI

摘要： 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-1 alpha) site of Bxj genome by cross-over homologous recombination technique. Twenty mu g 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.

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