半胱氨酸蛋白酶基因在广州管圆线虫致病中的机制研究

Human angiostrongyliasis is an emerging foodborne parasitosis caused by Angiostrongylus cantonensis. Disease presents following the ingestion of third-stage larvae (L3) residing in the intermediate mollusk host and disease manifests as eosinophilic meningitis or meningoencephalitis. The ingested larvae penetrate into the intestinal blood vessels and eventually reach the central nervous system(CNS) where develop into fifth-stage larvae(L5). The pathogenicity and pathophysiology of cerebral angiostrongyliasis, however, still poorly defined, and no effective strategy to prevention and therapy. It is urgent for us to further study on pathogenesis. Research suggests that invading the CNS is the critical stage of development. Based on the cDNA library from fifth-stage larvae of A.cantonensis which we had constructed, we found that cysteine proteases are the key elements which secreted by fifth-stage larvae of A.cantonensis. Cysteine protease is a important molecule in parasite-host interaction, involving in a number of important functions such as virulence, tissue migration, nutritional uptake and the suppression of host immune responses. In this study, we prepare to clone two cysteine protease genes, cathepsin B and cathepsin L of A.cantonensis, and then study the immunological and biological characters of the purified recombinant proteins, discuss their role in parasite-host interaction, verified whether they are directly damage the blood-brain barrier, and explore the immunopathogenesis of angiostrongyliasis. Our study may contribute to eradicate A.cantonensis.

广州管圆线虫（Ac）是一种新现的食源性寄生虫，三期幼虫（L3）经口感染人体后最终侵入中枢神经系统发育为L5期幼虫，引起患者嗜酸性粒细胞增多性脑膜炎或脑膜脑炎，严重者可致死。然而到目前为止广州管圆线虫的致病机制仍未阐明，给疾病的防治带来困难，需进一步研究。我们前期构建了L5期幼虫的cDNA文库，经过系统分析表明，半胱氨酸蛋白酶类是广州管圆线虫L5期幼虫寄生脑部时分泌的重要蛋白酶。基于此点，本项目拟采用分子生物学、酶活性分析、细胞培养及动物感染模型等手段，详细研究Ac半胱氨酸蛋白酶在虫体移行至脑部致病中的作用。具体内容包括对半胱氨酸蛋白酶基因AcCPB2及AcCPL进行重组表达并获得活性蛋白；利用酶活性分析及蛋白水解实验阐明其在虫体与宿主相互关系中的作用，验证半胱氨酸蛋白酶是否直接破坏宿主血脑屏障；并探究其致病的可能免疫机制，加深我们对广州管圆线虫致脑损伤机制的了解，为该病的防治提供理论基础。