应用Label-free技术筛选日本血吸虫肺期童虫差异表达体被被膜蛋白

Compared with other developmental stages of schistosome in the final host, the lung-stage schistosomulum is more vulnerable to the host immune system. The tegument-exposed proteins not only play important roles in nutrition intake, immune evasion, excretion and signal transduction for schistosome, but also could induce immunity reaction of the host immune system. The differentially expressed tegument-exposed proteins of lung-stage schistosomulum are likely to be important protective antigens. In this study, a streptavidin-biotin affinity purification technique will be used to isolate tegument-exposed proteins of lung schistosomulum and adult worm of Schistosoma japonicum, which will be then subjected to label-free quantitative proteomics and bioinformatics analyses to screen the pivotally, differentially expressed tegument-exposed proteins of lung schistosomulum. For these pivotal tegument-exposed proteins, transcriptional levels of their genes at different developmental stages of schistosome will be studied by Real-time PCR. Their distribution in worms will be observed by immunofluorescence. Antigenicity of their recombinant proteins will be analyzed by western blotting. The immune protective effect against schistosomiasis induced by their recombinant proteins will be assessed in the mouse model. Our study will provide the theoretical basises for the clarification of growth and development mechanisms of S. japonicum in the final host and the screening of vaccine candidates against schistosomiasis.

血吸虫肺期童虫是最易被宿主免疫系统攻击的发育阶段。体被被膜蛋白不仅在血吸虫营养摄取、免疫逃避、排泄和信号转导等方面扮演着重要角色，更是诱导宿主免疫系统发生免疫反应的重要分子，其在肺期童虫发育阶段差异表达的体被被膜蛋白极有可能是重要的保护性抗原靶标。本项目拟应用生物素-链酶亲和素纯化技术提取日本血吸虫肺期童虫和成虫的体被被膜蛋白，利用非标记（Label-free）定量蛋白质组学和生物信息学技术对其进行系统的比较分析，筛选肺期童虫差异表达的关键体被被膜蛋白，利用Real-time PCR技术分析其编码基因在不同发育阶段虫体的转录水平，采用免疫荧光染色实验分析蛋白在虫体的组织分布，应用Western blotting技术检测重组蛋白的抗原性，并通过小鼠免疫保护试验评估它们在抗日本血吸虫感染中的价值，从而为阐明日本血吸虫在终末宿主体内的生长发育机制及筛选抗血吸虫病疫苗候选抗原分子提供理论依据。

日本血吸虫肺期童虫被认为是免疫效应机制的主要靶点，而其体被结构是免疫效应分子攻击虫体的直接靶标，因此，肺期童虫差异表达蛋白（尤其是体被被膜蛋白）极有可能是重要的保护性抗原候选分子。本研究以3d肺期童虫为研究对象，42d成虫为对照，利用Label-free非标记定量蛋白质组学、Shotgun定性蛋白质组学技术分别鉴定了两个时期虫体的全虫蛋白、体被被膜蛋白，并结合生物信息学技术对其进行系统的比较分析。全虫蛋白的分析表明，3d肺期童虫差异表达蛋白共629个，其中上调表达194个、下调表达435个。GO功能富集分析获得20个显著性富集的GO条目，大量与单生物体过程、催化活性、结构分子活性等相关的蛋白表现为富集。KEGG通路富集分析表明，差异表达蛋白主要与转录、运输和分解代谢、碳水化合物代谢、氨基酸代谢、蛋白质的折叠与降解等过程有关。PRM技术验证结果显示，参与定量的10种蛋白表达趋势与蛋白组学结果基本一致。体被被膜蛋白的分析筛选到包括抗增殖蛋白（Prohibitin，PHB）两个亚型在内的部分肺期童虫高表达体被被膜蛋白。利用PCR技术获得SjPHB1（825bp）、SjPHB2（900bp）的ORF序列，并构建重组表达质粒pET-32a(+)-SjPHB1、pGEX-4T-1-SjPHB2，二者均在大肠杆菌系统中以包涵体形式获得表达与纯化，重组蛋白的理论分子量约为47kDa、59kDa。Western blotting 分析表明，rSjPHB1和rSjPHB2具有较好的免疫原性。应用纯化的rSjPHB1和rSjPHB2分别结合ISA206佐剂免疫小鼠，虽然针对两个重组蛋白的特异性IgG抗体滴度显著升高，但二者未能诱导产生显著的免疫保护效果。免疫荧光染色试验分析显示，SjPHB1和SjPHB2在虫体体被上有明显重合。qPCR结果表明，SjPHB1和SjPHB2在虫体不同发育阶段均有表达，在总体趋势上，两个基因在童虫的转录水平高于成虫、雌虫的转录水平高于雄虫，而在成虫的转录水平又高于虫卵、毛蚴、尾蚴。体内外的RNA干扰实验表明，siRNA727/796组合干扰虫体时，SjPHB1和SjPHB2转录水平降低最显著，不仅诱导小鼠获得31.35%的减虫率，而且可致雌雄虫体表结构被破坏。研究结果为阐明日本血吸虫在终末宿主体内的生长发育机制及筛选抗血吸虫病疫苗候选抗原分子提供了理论依据。