Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites
Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites
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通过第二代裂殖子核转染稳定转染恶毒艾美耳球虫
DOI：
10.1016/j.molbiopara.2019.01.002
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发表时间：
2019-03-01
影响因子：
1.5
通讯作者：
Liu, Xianyong
中科院分区：
文献类型：
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作者：
Duan, Chunhui;Hu, Dandan;Liu, Xianyong
艾美球虫属，球虫病的病原体是鸡最常见的原生动物病原体。感染这些寄生虫可导致动物发育不良或死亡，从而对家禽生产造成毁灭性的经济影响。建立艾美球虫属物种的遗传操作和基因的稳定表达的转染方案将有助于推进这些寄生虫的生物学，以及建立这些生物体作为新的疫苗递送载体。在此，我们报道了第一个稳定的转基因大肠杆菌的筛选。necatrix种群EnHA1，在用含有EYFP报告基因、来自禽流感病毒H9N2的HA1基因和TgDHFR-TS选择标记的线性DNA片段转染第二代裂殖子后，组成性表达EYFP报告基因，所述选择标记赋予对乙胺嘧啶的抗性。将转染的裂殖子通过泄殖腔途径接种到鸡体内，收集接种后18 h至72 h的粪便，并进行后续连续传代、流式细胞仪分选和乙胺嘧啶选择。EYFP表达孢子化卵囊的数量逐渐增加，在5次传代后获得超过90%的EYFP+卵囊。免疫荧光测定证实了HA1抗原在EnHA1群体中的成功表达。基因操纵E. necatrix裂殖子并在该寄生虫中表达异源基因将为该生物体作为疫苗递送载体的可能用途铺平道路。
Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle.