吉陶单极虫分泌蛋白酶作用鱼寄主的分子机制

In view of the situations for Myxosporean, such as frequent occurrence of diseases, difficult to control, great harm and heavy losses, this project will focus on Thelohanellus kitauei, one kind of Myxosporean which do great harm to intensive culture of carp. Based on previous study especially our genomics study, protease was shown to play a key role in tissue invasion and nutrition metabolism of Myxosporean. The predicted 14 secretory proteases of T. kitauei genome will be investigated, and the key secretory proteases, which are high-expression or high differential expression in the plasmodia and spore, will be screened by RT qPCR. High-activity secretory proteases will be screened by activity analysis. The distribution of these key secretory proteases in the T. kitauei or the infected tissues of carps will be investigated by Western blotting, immunohistochemistry and confocal immunofluorescence microscopy analysis. Host target proteins of secretory proteases will be determined by iTRAQ-TAILS. The degradation mechanism of host target proteins by secretory protease will be revealed by mass spectrometry of protease-digested productions. The function of host target proteins will be predicted by bioinformatics. The functions of host target proteins on cellular levels will be disclosed by RNA interferes and the transcriptional and proteomic analysis. The molecular mechanism of interaction between T. kitauei secretory protease and fish host will be elucidated. These research results will provide the basis for the in-depth study of host-parasite relationships, drug target validation and the new drug development against T. kitauei, which will promote healthy development in aquaculture.

针对粘孢子虫病害频发、难防治、危害大损失重现状，从鲤鱼集约化养殖危害严重的吉陶单极虫病出发，在前期报道和我们组学研究表明蛋白酶在粘孢子虫组织入侵和营养代谢中重要性大的基础上，以14个预测的吉陶单极虫可分泌蛋白酶为研究对象，实时定量PCR筛选在营养体和孢子中高表达或表达差异大的可分泌蛋白酶，酶学性质研究分析高活性的可分泌蛋白酶，通过免疫印迹、免疫组化和免疫荧光共聚焦锁定在虫体表面或虫体外有分布的关键分泌蛋白酶；采用iTRAQ-TAILS定量N端蛋白组学方法鉴定关键分泌蛋白酶作用鱼寄主的寄主靶蛋白，通过酶解靶蛋白产物的质谱分析揭示分泌蛋白酶降解寄主靶蛋白机制；生物信息学预测靶蛋白功能， RNA干扰寄主靶蛋白结合转录组和蛋白组分析，在细胞水平上解析寄主靶蛋白所涉及的功能，阐明吉陶单极虫分泌蛋白酶作用鱼寄主的分子机制，为深入寄生虫与宿主互作研究、确证药靶及新药开发奠定基础，促进水产健康养殖。

粘孢子虫病是一种对渔业生产造成严重危害的寄生虫病害。粘孢子虫与鱼寄主的作用机制不明限制了其防治药物的开发。本研究从前期吉陶单极虫基因组研究预测的与营养代谢和入侵相关的可分泌蛋白酶和可分泌蛋白酶抑制剂出发，表达水平分析表明预测的53个可分泌蛋白酶及蛋白酶抑制剂中有37个基因可实现扩增，有7个基因在孢子阶段表达水平较高，有4个基因扩增序列正确且实现了异源表达，其中TK-pepsin编码天冬氨酸蛋白酶，TK-papain-1和TK-papain-2编码半胱氨酸蛋白酶，TK-cystatin编码半胱氨酸蛋白酶抑制剂，进化树分析发现TK-pepsin和TK-cystatin分别与水螅蛋白酶和蛋白酶抑制剂的亲缘关系较近，进一步支持粘孢子虫属于刺胞动物门。对实现可溶性表达的TK-cystatin进行了深入研究。活性研究表明TK-cystatin对蛋白酶papain、人cathepsin L、人cathepsin B均有抑制活性，对人cathepsin L的抑制活性最强。免疫荧光定位结果表明TK-cystatin蛋白主要位于粘孢子虫钝端的胚核附近。TK-cystatin能引起巨噬细胞活性的降低，暗示其具有抑制宿主细胞对吉陶单极虫的免疫反应。Pulldown 实验发现和验证了TK-cystatin与鲤鱼CC cathepsin L蛋白存在相互作用。分子对接分析表明TK-cystatin通过氢键和盐键与鲤鱼CC cathepsin L表面形成相互作用，两个蛋白的结合能为−10.6 kcal/mol。功能预测表明鲤鱼CC cathepsin L具有调节细胞凋亡和免疫等功能。以上研究表明吉陶单极虫可以通过其分泌的蛋白酶抑制剂TK-cystatin抑制宿主鲤鱼的cathepsin L活性，从而降低宿主对吉陶单极虫的免疫反应。本研究为粘孢子虫病防治药物开发奠定了基础。

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