Establishment of a transient transfection system for Babesia sp. Xinjiang using homologous promoters
Establishment of a transient transfection system for Babesia sp. Xinjiang using homologous promoters
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巴贝虫瞬时转染系统的建立。
DOI：
10.1007/s00436-021-07250-4
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发表时间：
2021
影响因子：
2
通讯作者：
H. Yin
中科院分区：
文献类型：
--
作者：
Xiaoxing Wang;Jinming Wang;Jun Liu;Jifei Yang;Z. Lv;Ai;You;Yubin Li;Haiou Lan;Guangyuan Liu;Jian;Guiquan Guan;H. Yin
巴贝斯虫是人类和动物巴贝斯虫病的主要病原体，广泛分布于世界各地。在过去的十年里，基因操作方法已经被应用于许多原生动物寄生虫，包括恶性疟原虫、克氏锥虫、微小隐孢子虫、环形泰勒虫、泰勒氏虫、牛巴贝斯虫、双线巴贝斯虫、卵形巴贝斯虫、吉氏巴贝斯虫和绵羊巴贝斯虫。对于Babesiasp来说。新疆绵羊巴贝斯虫病主要病原为新疆巴贝斯虫病，主要发生在中国，但这些技术的效果尚不清楚。首先，通过电穿孔和核孔法将含有延长因子-1α启动子、萤火虫荧光素酶报告基因和RAP停止区的质粒导入BspXJ，确定最适的转染液。然后，对六种程序设置进行了评估，确定了BspXJ瞬时转染的最佳方案，并对一系列不同量的质粒DNA进行了转染，以产生相对较高的发光值。最后，利用所建立的瞬时转染系统对BspXJ来源的四个启动子的活性进行了评价。通过对各种转导参数的比较，确定了人T细胞核受体溶液、程序V-024和20μg为BspXJ瞬时转导的最佳条件。这些发现为BspXJ的基因操作提供了关键信息，BspXJ是识别毒力因子和进一步阐明这种寄生虫的基本生物学的重要工具。
Babesiaspecies, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, includingPlasmodium falciparum,Trypanosoma cruzi,Cryptosporidium parvum,Theileria annulata,Theileria parva,Babesia bovis,Babesia bigemina,Babesia ovata,Babesia gibsoni, andBabesia ovis. ForBabesiasp. Xinjiang (BspXJ), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remains unclear. Firstly, a plasmid bearing the elongation factor-1 alpha promoter and the firefly luciferase reporter gene and rap stop region were transfected into BspXJ by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program settings were evaluated to confirm the best for BspXJ transient transfection, and a series of different amounts of plasmid DNA were transfected to generate relatively high luminescence values. Finally, the activities of four promoters derived from BspXJ were evaluated using the developed transient transfection system. After evaluating of various transfection parameters, the human T cell nucleofector solution, program V-024 and 20 μg of plasmid DNA were selected as the most favorable conditions for BspXJ transient transfection. These findings provide critical information for BspXJ genetic manipulation, an essential tool to identify virulence factors and to further elucidate the basic biology of this parasite.