【作者】 陆婕；

【导师】 陈正望；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 家蝇（Musca domestica）属昆虫纲,双翅目,环裂亚目、蝇科,是我国大部分地区最常见、数量最多的一种蝇类。家蝇中存在大量抗菌活性物质,对抗菌药物的开发具有重要的意义。但家蝇中抗菌肽的种类较多,成分复杂,很难分离纯化出一个单一抗菌肽,目前国内外对其研究较少。本研究从家蝇干蝇蛆中分离纯化出了一个新抗菌肽Muscatoxin并测得其氨基酸序列,也深入开展了生理功能的研究。完成的工作主要包括以下几个方面:1、利用短时沸水处理、稀醋酸低温浸提、海藻酸吸附、稀盐酸低温洗脱、盐析、凝胶过滤、CMC23弱阳离子交换柱层析、反相高效液相色谱层析等方法,从家蝇蛆中分离纯化出一组蝇蛆抗菌肽,具有抗菌谱广,最低杀菌浓度低,耐热、耐冻融能力极强等特点。其杀菌活性高低依次为:枯草杆菌＞苏云金芽胞杆菌＞金黄色葡萄球菌＞铜绿假单孢菌＞大肠杆菌,最低杀菌浓度依次为:0.039、0.078、1.25、5.0、20.0μg/μl。蝇蛆抗菌肽不是凝集素类物质,对G⁺菌的杀灭作用比对G^-菌强,对抗菌药物的开发有重要意义。此外,蝇蛆抗菌肽中存在的大量弱酸性蛋白也有杀菌活性。2、结合反相高效液相色谱层析、聚丙烯酰胺凝胶电泳和电洗脱回收等方法,首次在蛋白质水平上,从蝇蛆抗菌肽中分离纯化出一个高纯度的新抗菌肽Muscatoxin,分子量7095 D,等电点8.88,肽质量指纹谱（PMF）分析表明其为一新肽,Edman降解测得其N端40个氨基酸序列为SQLGDLGSGA GKGGGGGGSI REAGGAFGKLEAAREEEYFY,N端有六联体的Gly、二联体的Gly和三联体的Glu结构域,可归类于富含Gly的抗菌肽。Muscatoxin可能存在蛋白质翻译后修饰。3、经GeneBank数据库查询,Muscatoxin是一个未曾报道过的新肽,其基因在原核生物和真核生物中都有。此序列与基因库中由果蝇Drosophila melanogaster的NM₁66597和AY071315基因推导拟蛋白的24～63aa同源性达97.5%,序列高度一致,仅第三位Val变成了Leu（这两种氨基酸的疏水性质很相似）:果蝇中该拟蛋白的序列是从基因序列推导而来的,我们首次从蛋白质水平上分离纯化出了该多肽。4、采用扫描电镜技术,发现蝇蛆抗菌肽Muscatoxin能破坏G⁺菌（枯草杆菌、苏云金芽胞杆菌、金黄色葡萄球菌）和G^-菌（铜绿假单孢菌、大肠杆菌）的细胞膜,使其形成穿孔,原生质泄漏而死亡;5、蝇蛆抗菌肽Muscatoxin能杀灭单核白血病癌细胞（THP-1）。经Muscatoxin（2mg/ml）处理后,白血病癌细胞的贴壁性很快被破坏,大量细胞悬浮并死亡。扫描电镜观察发现,悬浮细胞的细胞间质明显减少,细胞形态发生变化,细胞膜出现大量穿孔、原生质泄漏,细胞破裂死亡。Muscatoxin对癌细胞和细菌的杀灭机制相同:6、蝇蛆抗菌肽Muscatoxin有一定的哺乳动物细胞毒性。在高浓度（2 mg/ml）情况下能破坏小鼠全部血细胞的细胞膜,而在低浓度（0.08 mg/ml）情况下仅杀死白细胞,对小鼠的红细胞影响不大,但红细胞的形态变得更圆;7、采用膜片钳技术,发现mM级浓度的蝇蛆抗菌肽Muscatoxin能在胰腺β-细胞上形成阳离子通道,而在原生受体和离子通道少的人胚胎肾细胞（HEK293）上不能形成通道。说明Muscatoxin能与β-细胞上特定的受体相互作用后再使细胞膜穿孔形成离子通道,而且这种穿孔效应不依赖于胞外Ca²⁺的作用;8、采用胞内荧光测钙技术,发现mM级浓度的Muscatoxin使胰腺β-细胞膜穿孔后外钙内流,使得胞内钙浓度升高,这种作用与Muscatoxin的剂量无关,具有累积效应。Muscatoxin的作用浓度降低到fM级后仍有明显的效果,Muscatoxin对细胞膜的穿孔作用很强。9、从黑果蝇的蝇蛆中分离纯化出另一个新抗菌肽SK84,并测出其全部蛋白序列。SK84与Muscatoxin属于同一抗菌肽家族,并与其它种类的果蝇假蛋白有很高的同源性。SK84在1-100μM范围内无溶血活性。SK84对单核白血病（THP-1）、肝癌（HepG2）、乳腺癌（MCF-7）等细胞有抑制作用,但对人胚胎肾细胞（HEK293）、仓鼠卵巢（CHO）细胞无影响。更多还原

【Abstract】 Musca domestica, which belongs to insecta, diptera, cyclorrhapha, muscidae, is the most common muscae and the richest resource. It is very significant and valuable to isolate antibacterial peptides from Musca domestica and to develop these peptides into antibacterial medicine. Due to purify a pure peptide from the natural materials （animal, plant and microorganism tissue） is very difficult and complex, few research is going on. For the first time, we have purified a novel antibacterial peptide and designed as Muscatoxin based on its bioactivity and resource from larvae of Musca domestica. We have also chemically and biologically characterized the peptide Muscatoxin.1、A group of antibacterial peptides had been isolated from larvae of Musca domestica by a number of batch-wise biochemistry separation followed by several chromatography steps. The antibacterial peptides had characters of broad antibacterial spectrum、low minimum bactericidal concentration （MBC）、high thermostable and freeze-thaw stable. The bactericidal ability order was Bacillus subtilis> Bacillus thuringiensis> Staphylococcus aureus> Pseudomonas aeruginosa> Escherichia coli. and the MBC is 0.039、0.078、1.25、5.0 and 20μg/μl respectively. It is significant to develop antibacterial medicine for that the peptides are not agglutinin and have stronger activity to G⁺ than G^-. Furthermore, a plenty of weakly acid protein in the peptides also had bactericidal activity.2、Using reverse-phase HPLC、polyacrylamide gel electrophoresis and electroelution, the novel antibacterial peptide Muscatoxin had been purified in high degree of purity. It was determined pI 8.88 with IEF-PAGE, Mr 7095 D with MALDI-TOF-MS spectrometer. PMF showed Muscatoxin was a novel peptide and the N-terminal amino acid sequence was SQLGD LGSGA GKGGG GGGSI REAGG AFGKL EAARE EEYFY. Muscatoxin was belong to glysine-rich peptides with （Gly）₆、（Gly）₂ and （Glu）₃ domains and maybe had been post-translation modified.3、Searching in Genebank database, Muscatoxin was a novel peptide which had not been reported. Muscatoxin was 97.5% homologenous with pseudoprotein of Drosophila melanogaster （NM₁66597 and AY071315, 24～63aa） from fruit fly gene bank except the third residue Leu vs. Val.4、Obseved by scanning electric mirror （SEM）, antibacterial peptide Muscatoxin could destroy the cell membrane of G⁺ （Bacillus subtilis、Bacillus thuringiensis、Staphylococcus aureus） and G^- （Pseudomonas aeruginosa、Escherichia coli. ） bacterium. The mechanism was to perforate the cell membrane and lead to bacterium lysis and die;5、Muscatoxin could kill mononuclear leukaemia cancer cells （THP-1）. After treated by Muscatoxin （ 2mg/ml ） , most leukaemia cells suspended for the destroyed anchorage-dependent characterization and died. Obseved by SEM, the intercellular substance decreased obviously and cell morphology had changed. The mechanism was also to perforate the cell membrane and lead to cancer cells lysis and die;6、Muscatoxin showed certain cytotoxicity to mammalian cells. In high concentration （2mg/ml）, it could totally lysis all blood cells membrane; but in low concentration （0.08 mg/ml）, it just killed leucocyte and had few effect on rat akaryocyte except more round morphology;7、Using patch clamp technique, it was found that Muscatoxin in mM could form cationic channels in pancreas 3-cells but not in human embryo-kidney cells （HEK293） which had few innate receptors and ion channels. It was suggested that Muscatoxin could interact with certain receptors inβ-cells and then perforated the cells membrane to form ion channels. The perforating effect did not depend on extracellular Ca²⁺;8、Using intracellular Ca²⁺ fluorimetry technique, it was found that Muscatoxin in mM could perforateβ-cells and make the extracellular Ca²⁺ flow inwardly to increase the intracellular Ca²⁺ concentration. The effect is cumulative and not related to Muscatoxin dose. It was also apparently effective even the Muscatoxin concentration decreased to fM.9、Another novel antibacterial peptide named SK84 was purified from larvae ofDrosophila virilis and characterized total protein sequence. SK84 had no haemolytic activity from 1 to 100μM. SK84 could inhibite THP-1、HepG2 and MCF-7 cancer cells, but not affect HEK293 and CHO cells.更多还原