基于转录组学和多肽组学的金黄虎头蜂蜂毒中钠离子通道选择性镇痛肽的系统挖掘及活性评价

Chronic pain is called an undead cancer. However, there is few high-efficiency and low-toxic analgesics for clinical use. Thus, it is important to find new analgesic candidates. Based on the traditional use of wasp venomic wine for the treatment of rheumatoid arthritis among the Jingpos, our previous work have showed that the peptides in venom exhibit obvious analgesic and anti-inflammatory activity. However, some challenges were encounted in further research, including the limited resources of wasp venom, low content of active ingredients, as well as the unknown targets. Therefore, a transcriptome of venom gland and peptideomics of its venom were performed to systematically describe the peptides in venom of Vespa mandarinia. The inflammatory pain related sodium channel NaV1.7 was further selected for virtual screening to identify high-affinity peptide ligands in venom. Finally, solid phase peptide synthesis was used to provide sample of trace peptides, and the analgesic activity and toxicity of lead peptides were evaluated on cellular and animal levels. The results of this study will not only enrich the understanding of the peptides in wasp venom, but also provide lead compounds for the development of analgesics selectively toward sodium channel. Moreover, the method used in this research which combining transcriptomics, peptideomics and solid phase peptide synthesis for the discovery of trace peptides, and the technology used in the discovery of target-selective peptide ligands in animal drugs will provide new choice for the development of rare animal medicinal materials.

慢性疼痛被称为不死的癌症，然而临床缺乏高效低毒的镇痛药，寻找新的镇痛物质具有重要意义。基于景颇族采用蜂毒酒来治疗类风湿性关节炎的传统，本团队前期研究发现胡蜂毒液中多肽具有明显的镇痛抗炎活性。然而进一步研究面临蜂毒资源有限，活性成分含量低，作用靶点未知等挑战。基于此本研究以金黄虎头蜂为对象，拟通过毒腺转录组学和毒液多肽组学的研究，系统阐述毒液的多肽组成。进一步选择与炎性疼痛相关的镇痛靶点NaV1.7进行虚拟筛选，发掘蜂毒多肽中钠离子通道高亲和力配体。最后采用多肽固相合成技术解决微量多肽来源问题，并从细胞、动物水平上评估先导肽的镇痛活性及毒性。本研究的开展不仅将丰富对胡蜂蜂毒多肽的认识，并为钠离子通道选择性镇痛药的开发提供先导化合物。此外该研究构建的结合转录组学、多肽组学和多肽固相合成的微量多肽发掘模式，和动物药中靶点选择性多肽配体发现的研究体系将为稀有动物药材的现代化开发提供新的思路和选择。

慢性疼痛被称为不死的癌症，然而临床缺乏高效低毒的镇痛药，寻找新的镇痛物质具有重要意义。基于景颇族采用蜂毒酒来治疗类风湿性关节炎的传统，本团队前期研究发现胡蜂毒液中多肽具有明显的镇痛抗炎活性。然而进一步研究面临蜂毒资源有限，活性成分含量低，作用靶点未知等挑战。基于此本项目完成了以下工作①采用转录组学相关技术，通过解剖金黄虎头蜂分离毒腺并提取mRNA，进行测序、翻译得到129213条转录本；通过功能注释从GO数据库阐述了其中21445条转录本的功能、KEGG数据库注释了其中17402条转录本生物功能、NR数据库注释了其中31605条转录本生物功能、PFAM数据库注释了其中18337条转录本生物功能、STRING数据库注释了其中1727条转录本生物功能、SWISSPROT数据库注释了其中21929条转录本生物功能。进一步的生信分析从中获得8298条蛋白多肽序列（ORF）。②通过采集金黄虎头蜂毒液，并进行电泳分析发现抗原5、透明质酸酶和磷脂酶PLA1是蜂毒中主要的过敏原；质谱分析显示其中86%的多肽分子量在300–3000Da范围内；进一步的多肽组学研究，从中得到39个蛋白质和167条多肽序列；③完成所得多肽库中NaV1.7选择性阻断剂的虚拟筛选，选择其中12条进行多肽固相合成，细胞和动物水平活性测试显示其中1条多肽镇痛活性与阳性药相当。本项目的工作不仅丰富了对胡蜂蜂毒多肽的认识，并为钠离子通道选择性镇痛药的开发提供先导化合物。此外该研究构建的结合转录组学、多肽组学和多肽固相合成的微量多肽发掘模式，和动物药中靶点选择性多肽配体发现的研究体系将为稀有动物药材的现代化开发提供新的思路和选择。