哈氏蜈蚣Scolopendra subspinipes dehaani毒素组学和转录组学研究
文献类型：学位论文
| 作者 | 刘子超 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 蜈蚣 毒素 cDNA 文库 转录组学 蛋白质组学 |
| 其他题名 | Venomic and Transcriptomic Analysis of Centipede Scolopendra subspinipes dehaani |
| 学位专业 | 动物学 |
| 中文摘要 | 蜈蚣是一类古老的陆生无脊椎动物，在全球广泛分布。作为一味传统中药材，蜈蚣在临床上应用已经有两千多年历史。蜈蚣的第一对步足特化为颚肢，能分泌毒液，是蜈蚣捕食和防御的主要器官。蜈蚣毒液的成分十分复杂，其中很多具有不同的生化和药理学特性。然而，到目前为止，人们对蜈蚣毒素的认识仍然十分有限，蜈蚣毒素成分还没有真正系统地被研究过。哈氏蜈蚣Scolopendra subspinipes dehaani在中国海南、广东、广西、云南一带广泛分布，其个体大，毒液量多，容易捕捉，适合用于毒素研究。本论文从转录组学、蛋白质组学和生物学功能三个层面对哈氏蜈蚣毒素进行研究。在转录组学层面：通过构建cDNA文库和随机选择大约1 500个独立克隆测序，得到1 122个全长cDNA序列，它们编码543个不同的蛋白质序列，转录组学分析表明这些序列可以分为三类：无提示类（no hits）有308条序列，占56.72%；分泌蛋白/多肽(secreted proteins/peptides)类有139条，占25.60%；细胞组分(cellular components)类有96条，占17.68%。在分泌蛋白/多肽类序列中，神经毒素、离子通道活性成分和毒液过敏原占绝大多数。蛋白质组学层面：通过Sephadex G 50凝胶过滤、?KTA Resource Q阴离子交换柱、?KTA Mono Q阴离子交换柱、RP-HPLC C4柱、RP-HPLC C18柱等分离纯化过程，从哈氏蜈蚣毒液中得到55个经过N-末端测序和质谱分析的蛋白质/多肽，去除相同组分在不同分离峰中出现的情况，得到40个不同的蛋白质/多肽，其中29个蛋白质/多肽与测定的cDNA序列完全匹配。生物学功能层面：对从哈氏蜈蚣粗毒中分离得到的蛋白质/多肽组分进行多项生物学功能检测，共检测到下列活性：（1）1个组分具有血小板聚集活性和溶血活性；（2）1个组分具有抗凝活性；（3）2个组分具有磷脂酶A2活性；（4）1个组分具有胰蛋白酶抑制剂活性；（5）9个组分具有电压门控型钾离子通道抑制活性；（6）1个组分具有电压门控型钠离子通道抑制活性和（7）4个组分具有电压门控型钙离子通道抑制活性。在哈氏蜈蚣粗毒中检测到出血活性，但是在各组分中没有检测到出血活性；没有检测到抗菌活性和明显的细胞毒性。与已知数据库比对结果表明，这些蛋白质/多肽中大多数序列与目前已知序列无显著相似性，表明与其它有毒动物类群相比，蜈蚣毒液的成分有其新颖性。本研究第一次从转录组学、蛋白质组学和生物学功能三个层面对蜈蚣毒素进行了较为系统的研究，提供了目前为止数量最大的蜈蚣毒素成分，对哈氏蜈蚣毒素的本质起到一定的揭示作用。 |
| 英文摘要 | entipedes are a class of ancient terrestrial invertebrates widely distributed in the world. They have been used as traditional medicines with great clinical importance for more than two thousand years. Centipedes have venom glands in their first pair of limbs which is the most important organ for predation and defense. The venoms secreted by these glands contain a large number of components with different biochemical and pharmacological properties. However, information about the compositions and functions of their venoms is largely unknown. Scolopendra subspinipes dehaani is widely distributed in Hainan, Guangxi, Guangdong and Yunnan provinces. Its body is very large. So, it can provide enough venom for studying. In this study, Scolopendra subspinipes dehaani venoms were investigated by transcriptomic and proteomic analysis coupled with biological function assays. After random screening approximately 1500 independent clones, 1122 full length cDNA sequences, which encoding 543 different proteins, were cloned from a constructed cDNA library using a pair of venom gland from a single centipede species. Based on the protein blast and functional assay results, these proteins/peptides were classified into no hits (308, 56.72%), secreted proteins/peptides (139, 25.60%) and cellular components (96, 17.68%), respectively. Neurotoxins, ion channel acting components and venom allergens were the main fractions of the crude venom as revealed by transcriptomic analysis. Meanwhile, 40 proteins/peptides were purified and characterized from crude venom of S. subspinipes dehaani. The N-terminal amino acid sequencing and mass spectrum results of 29 out of these 40 proteins or peptides matched well with their corresponding cDNAs. The purified proteins/peptides showed different biological properties, including: (1) one component with platelet aggregating activity; (2) one component with anticoagulant activity; (3) two components with phospholipase A2 activity; (4) one component with trypsin inhibiting activity; (5) nine components with voltage-gated potassium channel activities; (6) one component with voltage-gated sodium channel activities; and (7) four components with voltage-gated calcium channel activities. Most of them showed no significant similarity to other protein sequences deposited in the known public database. Scolopendra subspinipes dehaani venoms were systematically investigated by transcriptomic and proteomic analysis coupled with biological function assays. This work provides the largest number of protein or peptide candidates with medical-pharmaceutical significance and reveals the toxin nature of centipede S. subspinipes dehaani venom. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10159]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 刘子超. 哈氏蜈蚣Scolopendra subspinipes dehaani毒素组学和转录组学研究[D]. 北京. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：昆明动物研究所
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