Taheri Tahereh, Saberi Nik Hana, Seyed Negar, Doustdari Fatemeh, Etemadzadeh Mohammad-Hossein, Torkashvand Fatemeh, Rafati Sima
Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran.
Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran; Fars Science and Research Branch, Islamic Azad University, Shiraz, Iran.
Exp Parasitol. 2015 Mar;150:44-55. doi: 10.1016/j.exppara.2015.01.008. Epub 2015 Jan 28.
Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes.
由于缺乏准确且灵敏的工具来评估寄生虫血症水平，利什曼病的治疗或预防仍是全球面临的一项重大挑战。为了通过两个参数实时监测和追踪利什曼原虫感染，我们构建了稳定表达融合了增强型绿色荧光蛋白（EGFP）和萤火虫荧光素酶的双报告蛋白的转基因利什曼原虫。同时使用两个报告基因（egfp - luc）可提高检测/诊断的实验灵敏度，以及对寄生虫进行体外定量和对小鼠实时感染情况的监测。通过不同的特定工具，在哺乳动物宿主和前鞭毛体中均可检测和测量来自寄生虫的EGFP和LUC信号。在此，LUC蛋白比EGFP具有更高的灵敏度，因此通过生物发光可在疾病早期在足垫（注射部位）和淋巴结中检测到感染。这些结果表明：（1）定量报告基因EGFP和LUC均可同时用于体外和体内检测寄生虫血症；（2）在原鞭毛体中，萤火虫荧光素酶的灵敏度比EGFP高10倍。
