一种高效抗多子小瓜虫天然化合物的作用靶蛋白研究

Ichthyophthirius multifiliis is a ciliated parasite with a global distribution. It causes “white spot disease” of freshwater fish and results in a significant economic loss in the aquaculture industry. Parasiticides commonly used to control I. multifiliis have potential environmental and mammalian toxicities. Since malachite green has been banned for use in food fish due to its carcinogenic and teratogenic effects on humans, there are not therapeutants with both safe and efficacy in treating I. multifiliis. The traditional way for screening effectively antiparasitic therapeutants against I. multifiliis is at low efficiency. High throughput screening based on drug target is an efficient and convenient way to find new drug candidiates. However, no drug target was reported for I. multifiliis in previous studies. Cynatratoside-C isolated and purified form Cynanchum atratum by bioassay-guided fractionation is a safe and efficient natural compound to treat I. multifiliis. Therefore, it is necessary to find the target protein targeted by cynatratoside-C for more efficient active compounds against the target protein by means of high throughput screening. In order to get the drug target for treating I. multifiliis, the target protein of cynatratoside-C will be isolated and identified with small molecule affinity chromatography and LC-MS/MS. Then the drug target protein is confirmed by a series of molecular biology techniques. Finally, the location of target protein in I. multifiliis is detected by immunofluorescence assy. This work will solve the key scientific problem for illumination of the antiparasitic mechanism of cynatratoside-C against I. multifiliis and provide drug target protein for high throughput screening of a lot of anti-I. multifiliis drug candidates with similar function of cynatratoside-C.

多子小瓜虫引起鱼类“白点病”造成严重经济损失。曾经有效防治小瓜虫病的孔雀石绿和汞制剂毒性大、残留高，已被禁用。迫切需要开发安全高效的防治小瓜虫病药物。用传统方法筛选防治小瓜虫病药物的速度慢，而以药物靶标为导向的高通量筛选候选药物更为高效。但至今没有小瓜虫药物靶标的报道。本课题组前期研究发现了一种迄今为止抗多子小瓜虫活性最好且安全性高的植物活性化合物直立白薇苷C。如果找到直立白薇苷C在多子小瓜虫中的作用靶蛋白，再以该靶蛋白作为筛选更多具有相似功能的候选安全高效药物的靶标，将加快防治小瓜虫病药物研发的步伐。同时，也为开发直立白薇苷C成商业抗虫渔药奠定坚实理论基础。本项目拟在前期工作基础上，分离鉴定直立白薇苷C在小瓜虫中的作用靶蛋白，明确其在虫体中的分布，为阐明直立白薇苷C杀虫分子机理解决关键科学问题，同时也为高通量筛选杀小瓜虫的候选药物提供药物靶标。

多子小瓜虫引起鱼类“白点病”造成严重经济损失。曾经有效防治小瓜虫病的孔雀石绿和汞制剂毒性大、残留高，已被禁用。迫切需要开发安全高效的防治小瓜虫病药物。用传统方法筛选防治小瓜虫病药物的速度慢，而以药物靶标为导向的高通量筛选候选药物更为高效。本课题组前期研究发现了一种迄今为止抗多子小瓜虫活性最好且安全性高的植物活性化合物直立白薇苷C。本项目旨在找到直立白薇苷C在多子小瓜虫中的作用靶蛋白，并阐明该蛋白在小瓜虫中的分布。采用小分子免疫亲和色谱技术富集和识别了直立白薇苷C在成虫中作用的靶蛋白，鉴定为ABC跨膜转运蛋白。通过RACE技术扩增ABC跨膜转运蛋白基因的全长cDNA为1924 bp，编码区长为1818 bp。采用原核表达技术分别获得了该蛋白膜内域和跨膜域膜外部分的重组蛋白，体外结合实验证明了直立白薇苷C能与膜内域结合。最后采用免疫荧光定位技术阐明了ABC跨膜转运蛋白位于小瓜虫成虫的细胞膜上。因此本项目不仅为阐明直立白薇苷C杀虫分子机理解决了关键问题，从而为开发该活性物成杀虫渔药奠定了坚实理论基础，而且为高通量快速筛选安全高效的抗小瓜虫候选药物提供了靶标，从而为建立新的高效药物筛选平台奠定了基础。