水蛭粘杆菌Mucinivorans hirudinis胞外血浆蛋白酶（MHP）促进摄血水蛭消化作用机制研究

水蛭及以其为主要原料的中成药是常用活血化瘀药，临床疗效十分显著，然而水蛭肠道内消化酶无法满足其消化需求，已成为人工养殖的瓶颈。摄血动物的消化问题一直是生物学领域研究的热点，水蛭肠道微生物在消化过程中起关键作用，但其机制尚不清楚。申请者前期研究发现水蛭摄血后肠道粘杆菌丰度急剧上升，且其胞外血浆蛋白酶（MHP）具有消化血浆蛋白的作用，推测MHP具有促进摄血水蛭消化的作用。据此，本研究拟采用全基因组和转录组技术筛选MHP基因、上游启动子及其转录因子，应用RT-PCR和免疫印迹法等方法研究MHP相关基因对水蛭粘杆菌、抗生素的表达响应，运用益生元、益生菌诱导MHP基因表达进行调控机制研究，借助自杀载体同源重组技术改造MHP基因进行基因功能验证。通过以上研究，探索MHP基因表达的信号通路和调控机制，阐明其促进摄血水蛭消化的作用机制，为水蛭乃至摄血动物的消化机制研究奠定理论基础，丰富和发展中药动物学。

Hirudo nipponica and its Chinese patent medicine are commonly used for promoting blood circulation and removing blood stasis, relying on their remarkable clinical efficacy. However, the digestive enzymes in H. nipponica intestine can not satisfy the demands of blood absorption and digestion, which has become the bottleneck of H. nipponica artificial culture. The digestive problems of bloodsucking animals have always been the research focus in the field of biology. The intestinal microorganisms of Hirudo nipponica play a key role in the digestion process, but its mechanism is still unclear. In the previous study, the applicant found that the abundance of intestinal Mucinivorans increased rapidly after Hirudo nipponica blood ingestion, and its extracellular plasmaproteinase (MHP) had the digestive ability. It is speculated that MHP can promote the blood ingestion and digestion in Hirudo nipponica. In this research proposal, MHP gene and its transcription factors will be screened by genome-wide and transcriptome technology. Gene cloning, RT-PCR and Western blotting will be used to study the expression response of MHP related genes to Mucinivorans hirudinis and antibiotics. The regulation mechanism of MHP gene expression induced by prebiotics and probiotics will be studied, and the gene function will be verified by modifying MHP gene with suicide vector homologous recombination technology. Through the above research, firstly, we explore the signal pathway and regulatory mechanism of MHP gene expression, then clarify the mechanism of MHP gene promoting the absorption and digestion of Hirudo nipponica, subsequently, lay a theoretical foundation for the study of absorption and digestion mechanism of Hirudo nipponica and even blood taking animals, and finally enrich and develop traditional Chinese medicine zoology.