【作者】 冯荦；

【导师】 肖杭；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2004， 硕士

【摘要】 第一部分：东亚钳蝎毒素2-2-5-5-1、2-2-5-6-1的分离纯化 东亚钳蝎多肽毒素中含有多种对离子通道有作用的活性多肽。本论文第一部分采用经典色谱法对东亚钳蝎毒素多肽进行了分离和纯化。首先依次使用Sephadex G-25体积排阻层析柱，Sephadex G-50体积排阻层析柱，CM Sephadex C-25离子交换柱，对粗毒素进行前期分离；然后采用反相高效液相(RP-HPLC)色谱柱对蝎毒素进行进一步的反复纯化。结果显示：由Sephadex G-25得到4个色谱峰，取峰2进行Sephadex G-50柱层析，得到3个峰：2-1，2-2，2-3；收集峰2-2，进行CM Sephadex C-25柱层析，得到5个峰：2-2-1，2-2-2，2-2-3，2-2-4，2-2-5；取峰2-2-5，进行进一步的RP-HPLC分离纯化，得到9个组分：2-2-5-1，2-2-5-2，2-2-5-3，2-2-5-4，2-2-5-5，2-2-5-6，2-2-5-7，2-2-5-8，2-2-5-9，再次用RP-HPLC进行纯化，得到单一组分2-2-5-5-1与2-2-5-6-1。南京医科人学顿士学位论文 第二部分:东亚钳蝎毒素2一2一5一5一1、2一2一5一6一l的鉴定 本论文第二部分研究所分离纯化的多肤的基本性质并对其进行了初步鉴定。(l)对多肤2一2一5一5与2一2一5一6质谱鉴定的结果表明，这两个峰均各含两种成分，峰2一2一5一5中的两种组分分子量分别为:72 1 4.36(即峰2一2一5一5一l)和7253.11，相对丰度为100:20.77:峰2一2一5一6中的两种组分分子量分别为: 7036.85(即峰2一2一5一6一l)和3180.69，相对丰度为100:30.03。(2)对峰2一2一5一5一1与2一2一5一6一1进行紫外扫描显示，这两种多肤均呈末端吸收。仅在ZO6nrn处左右吸光度较高。(3)对峰2一2一5一5一1氨基酸分析的结果提示其残基个数的大致比例:AsP:Thr:Ser:Glu:Gly:Ala:Val:Met:Ile:Leu:Tyr:Phe:Lys:His:Arg“4:l:8:4: 10:4:4:2:2:2:2:2:2:l:2。符合其肤链长度，且未发现与其氨基酸残基比例相近的东亚钳蝎多肤。(4)对多肤2一2一5一5与2一2一5一6进行Edman降解，结果未探测到信号峰，提示这两个多肤的N端被封闭。 第三部分:东亚钳蝎毒素纯化组分辣椒素受体镇痛活性初探 本论文第三部分对分离纯化的组分进行了辣椒素受体镇痛作用筛选的功能研究，结果表明:辣椒素受体筛选试验表明组分2一2一5呈阳性反应，说明其对胞浆钙离子有一定的激动作用，这种作用可能是辣椒素受体所介导的。全细胞膜片钳实验显示2一2一5一5与2一2一5一6对分别对K+、Na+通道有较明显的活性作用。提示蝎毒组分有进一步发掘成为新一类的镇痛药及离子通道工具药的潜力。更多还原

【Abstract】 Part I: The purification and isolation of Buthus martensi Karsch (BmK) ncurotoxin 2-2-5-5-1 and 2-2-5-6-1BmK neurotoxins 2-2-5-5-1 and 2-2-5-6-1 were purified from scorpion crude venom by classical chromatographic method. Sephadex G-25, Sephadex G-50 size exclusion column and CM Sephadex C-25 ion-exchange column were used to primarily purify the crude venom and then RP-HPLC was used for further purifying the venom. Four chromatographic peak were obtained with Sephadex G-25 column and 3 with Sephadex G-50 from the previously fraction 2. Peak 2-2 from the previously peak 2 were applied for CM Sephadex C-25 column. Five fractions were observed and peak 2-2-5 was applied to further purification with RP-HPLC. Within the 9 fractions from the former peak 2-2-5, fraction 2-2-5-5 and 2-2-5-6 displayed relative purification by HPLC chromatography. RP-HPLC procedure was employed to further purify these peaks. Eventually we secured purified fractions 2-2-5-5-1 and 2-2-5-6-1.Part II : The identification of peptides 2-2-5-5-1 and 2-2-5-6-1Study upon primary characterization and identification of these peptides referred to four parts as follows: (1) The result of ESI-MS showed that both the fractions 2-2-5-5 and 2-2-5-6 contain two components. The MW of the two components of 2-2-5-5 are 7214.36(i.e. 2-2-5-5-1) and 7253.11 respectively and the ratio of the relative abundance is 100:20.77. The MW of the two components of 2-2-5-6 are 7036.85(i.e. 2-2-5-6-1) and 3180.69 respectively and the ratio of the relative abundance is 100:30.03. (2) Both of 2-2-5-5-1 and 2-2-5-6-1 reveal end absorption by UV scan. The absorbency is the highest at 206nm or so. (3) Acid amino analysis was used to determinate the relative ratio of the acid amino residues: Asp: Thr: Ser: Glu: Gly: Ala: Val: Met: lie: Leu: Tyr: Phe: Lys: His: Arg 4: 1: 8: 4: 10: 4: 4: 2: 2: 2: 2: 2:2:1: 2. The sum of the number of the ratio is in agreement with the length of the peptide. The peptides of BmK that have a closely ratio to 2-2-5-5-1 were not found. (4) No signal was detected by Edman degradation. It is probably that these two peptides are N-terminal blocked.Part HI: The study upon analgesic activity of BmK purified fractions based on vanilloid receptor 1(VR1)The analgesic activity screen test based on VR1 showed that 2-2-5 has the positive activity to VR1, which indicated 2-2-5 probably has exciting activity on plasma calcium via VR1. Whole-cell patch clamp recording showed the fractions 2-2-5-5 and 2-2-5-6 have obviously effect on potassium and sodium channel respectively. All of the above demonstrated the potential of the peptides for analgesia and for tool drug upon ion channels.更多还原