Li Wei, Diao Yumei, Gong Pengtao, Suo Xun, Li Jianhua, Zhang Xichen
Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun 130062, China; College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, China.
Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun 130062, China.
Mol Biochem Parasitol. 2014 Mar-Apr;194(1-2):33-5. doi: 10.1016/j.molbiopara.2014.04.004. Epub 2014 Apr 21.
The apicomplexan protozoan Cryptosporidium parvum is an enteric parasite that affects a variety of mammal hosts including humans, and causes serious diarrheal disease in immunocompromised individuals, notably AIDS patients. Despite many advances in the development of transgenic techniques in many protozoan parasites over the past two decades, rare reports have been documented on the genetic manipulation on C. parvum. Achievement of the DNA-based transfection chiefly depends on the selection of an effective parasite genus-specific promoter. This report described the successful yellow (YFP-YFP) or red (RFP) fluorescent protein expression in oocysts and sporozoites of C. parvum controlled by the endogenous promoters of actin, alpha tubulin, and myosin genes using the restricted enzyme-mediated integration technique. One expression cassette in pBluescript backbone, YFP-YFP or RFP fused between 5' and 3' untranslated regions of actin gene, displayed the highest transfection efficiency with fluorescence rate around 50%. The established DNA-based transient transfection assay may contribute to a better understanding of the biology of Cryptosporidium species and their relationship with hosts and may also result in the development of more efficient molecule-based vaccines and drugs.
顶复门原生动物微小隐孢子虫是一种肠道寄生虫，可感染包括人类在内的多种哺乳动物宿主，并在免疫功能低下的个体（尤其是艾滋病患者）中引发严重的腹泻疾病。尽管在过去二十年中许多原生动物寄生虫的转基因技术取得了诸多进展，但关于微小隐孢子虫基因操作的报道却很少。基于DNA的转染成功主要取决于选择有效的寄生虫属特异性启动子。本报告描述了使用限制性酶介导整合技术，在肌动蛋白、α微管蛋白和肌球蛋白基因的内源性启动子控制下，微小隐孢子虫卵囊和子孢子中成功表达黄色（YFP - YFP）或红色（RFP）荧光蛋白。pBluescript骨架中的一个表达盒，即YFP - YFP或RFP融合在肌动蛋白基因的5'和3'非翻译区之间，显示出最高的转染效率，荧光率约为50%。所建立的基于DNA的瞬时转染试验可能有助于更好地理解隐孢子虫物种的生物学特性及其与宿主的关系，也可能导致开发更有效的基于分子的疫苗和药物。
