Stable transfection system for Babesia sp. Xinjiang.
Stable transfection system for Babesia sp. Xinjiang.
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DOI：
10.1186/s13071-021-04940-x
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发表时间：
2021-09-09
影响因子：
3.2
通讯作者：
Yin H
中科院分区：
文献类型：
--
作者：
Wang J;Wang X;Guan G;Yang J;Liu J;Liu A;Li Y;Luo J;Yin H
稳定的转染系统已经在许多原生动物寄生虫中描述，包括恶性疟原虫、隐孢子虫、牛巴虫、卵形巴虫和吉氏巴虫。新疆巴贝斯虫是绵羊巴贝斯虫病的病原菌，主要流行于中国各地，但这些技术的平台仍然缺乏。遗传操作技术是增强寄生虫生物学知识的有力工具，可能提供潜在的药物靶点和诊断标记。我们评价了杀稻瘟菌素（BSD）和WR 99210对Bxj的抑制效率。然后，构建了带有选择标记BSD、绿色荧光蛋白（GFP）基因和棒状体相关蛋白-1 3′终止子区（rap 3′ TR）的质粒。通过交叉同源重组技术将该质粒整合到Bxj基因组的延伸因子-1 α（ef-1α）位点。将20 μg质粒转染到Bxj裂殖子中。随后，在转染后24小时进行药物选择以产生转染的寄生虫。经转染、药物筛选和定殖后，成功获得了转染的寄生虫系Bxj-c1、Bxj-c2和Bxj-c3。PCR扩增和测序证实外源基因已整合到Bxj基因组中。免疫印迹（WB）和间接免疫荧光（IFA）检测结果表明，GFP-BSD已表达11个月。本研究成功地建立了稳定的Bxj转染系统。我们预计，这个平台将大大促进Bxj的基础研究。
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. 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. 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. 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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影响因子：
3.2
作者：
Liu M;Adjou Moumouni PF;Asada M;Hakimi H;Masatani T;Vudriko P;Lee SH;Kawazu SI;Yamagishi J;Xuan X
通讯作者：
Xuan X
影响因子：
2.6
作者：
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通讯作者：
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影响因子：
2
作者：
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通讯作者：
Li, X
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影响因子：
2.1
作者：
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通讯作者：
Yin, Hong
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影响因子：
2.6
作者：
Guan, Guiquan;Ma, Miling;Luo, Jianxun
通讯作者：
Luo, Jianxun