鸡寄生虫的成核和活体繁殖
Summary
在这里，我们提供了一种通过核化孢子菌或第二代美罗索特物实现鸡艾梅里亚寄生虫稳定转染的方法。表达异源抗原基因的转基因生物寄生虫可用作疫苗输送工具。
Abstract
转染是一个技术过程，通过它遗传物质，如DNA和双链RNA，被输送到细胞中，以修改感兴趣的基因。目前，转基因技术正在成为研究Eimeria（家禽和牲畜球菌病的病原体）不可缺少的工具。该协议提供了对美洲寄生虫稳定转染的详细描述：孢子石或第二代美罗索石的纯化和核化，以及转染寄生虫在体内传播。利用这个协议，我们实现了几种艾梅里亚的转染。总之，核素是一个有用的工具，以促进基因操作的人类寄生虫。
Introduction
Eimeria spp. 引起球菌病，给畜禽业带来巨大的经济损失。虽然抗凝球药物，并在一定程度上，衰减抗凝细胞疫苗，已被广泛用于控制球菌病，但仍存在缺陷，在耐药性，药物残留，和疫苗菌株的潜在扩散，重新获得毒性1。随着分子生物学的发展，转染已成为研究基因功能、研制新型疫苗、筛选Eimeria新药靶点的重要工具。
在过去的几十年中，转染已经成功地应用于疟原虫寄生虫，如疟原虫和弓形虫贡迪2，3，4，5，6。一项使用β-gal作为E.tenella转染报告员的研究在Eimeria7中试行了这种工作。E. tenella8、9、 E. mitis10和E. acervulina（张等人，未公开的数据）在鸡中成功转染。最近，我们通过核化11，利用E.necatrix的美罗佐特体实现了转染。
研究表明，表达异源抗原的Eimeria具有被开发为重组疫苗的潜力，如那些表达弯曲杆菌抗原A（CjaA）或鸡白莱金2（chIL-2）12，13。因此，该协议描述了对鸡中Eimeria spp的成核研究。该程序描述了孢子或美速物的纯化、用质粒DNA核化、克隆接种/静脉注射和体内繁殖，以帮助研究人员开始研究转基因Eimeria寄生虫。
Protocol
所有动物实验的鸡按照中国农业大学动物护理与使用委员会的指导方针进行饲养和维护，并遵循《涉及动物的生物医学研究国际指导原则》。实验得到了北京市实验室动物管理委员会的批准。 1. 提取和纯化艾梅里亚孢子石（例如，E. tenella） 释放孢子 在 2，300 x g处将二铬酸钾溶液中的孢子卵母细胞（2.5%，m/v）离心 1 x 107孢子卵母细胞…
Representative Results
该协议已用于转染美洲寄生虫。在这项研究中，图2A和图2B显示了E.necatrix的第二代梅龙特和美罗佐石，而图2C和图2D显示了使用密度梯度溶液后E.tenella的孢子和孢子石。还显示了在核化了麦罗索特或孢子石之后的…
Discussion
在20世纪90年代，为阿皮兰寄生虫开发了转染系统，并用于研究美洲寄生虫。最近，在E.tenella8，9和E.nieschulzi15进行了稳定的转染。通过转染第二代美罗佐特11，实现了E.necatrix的稳定转染。通过翼静脉接种E.acervulina的转染孢子球，解决了E.acervulina孢子菌无法通过氯气途径接种的药管（张…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家重点研究发展计划（2017YFD0501200）和国家自然科学基金（31572507、31772728和31873007）的支持。
Materials
| ATP-disodium | Sigma | A26209 | |
| Cellulose DE-52 | Solarbio | C8350 | |
| Constant Flow Pump | SHANGHAI JINGKE INDUSTRIAL CO., LTD. | HL-2B | |
| DMEM | MACGENE | CM15019 | |
| Glass beads | Sigma | Z250473-1PAK | |
| Glucose | Sigma | No. V900116 | |
| Glycine | Biotopped | G6200 | |
| HBSS | MACGENE | CC016 | |
| KH2PO4 | Sigma | No. V900041 | |
| Low Speed Centrifuge | BEIJING ERA BEILI CENTRIFUGE CO., LTD. | DT5-2 | |
| Magnetic Mixer | SCILOGEX | MS-H280-Pro | |
| MgCl2 | Sigma | 449164 | |
| MoFlo cell sorter | BeckMan Coulter, US | 201309995 | |
| NaHCO3 | Sigma | 144-55-8 | |
| Nucleofection device | LONZA/amaxa | 90900012 (Nucleofector II) | |
| PBS | Solarbio | P1010 | |
| Percoll (DG gradient stock solution) | GE Healthcare | 17-0891-09 | |
| Sodium taurodeoxycholate hydrate | Sigma | T0875 | |
| Sorvall Legend Micro 17 Microcentrifuge | ThermoFisher Scientific | 75002430 | |
| The composition of DMEM: 4.5 g/L glucose with sodium pyruvate, L-glutamine, and 25 mM HEPES. | |||
| Trypsin | Solarbio | T8150 | |
| Vortex Mixer | Beijing North TZ-Biotech Develop.co. | HQ-60-II | |
| Water Bath Thermostat | Grant Instruments (Cambridge), Ltd. | GD120,GM0815010 |
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Citazione di questo articolo
Duan, C., Tang, X., Hu, D., Zhang, S., Liu, J., Bi, F., Hao, Z., Suo, J., Yu, Y., Wang, M., Sun, P., Du, L., Suo, X., Liu, X. Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites. J. Vis. Exp. (156), e60552, doi:10.3791/60552 (2020).