【作者】 柏鸣；

【导师】 周立；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 本文首次从家蝇(Musca domestica)幼虫血淋巴中分离纯化到一种具抗菌活性的蛋白质，并对其部分理化和生物学性质进行了研究。通过体壁损伤法诱导家蝇幼虫产生免疫血淋巴，经沸水浴热变性、减压蒸馏浓缩、CM-Sepharose离子交换层析、Sephadex G-50凝胶过滤等步骤纯化后，聚丙烯酰胺凝胶电泳证明为单一蛋白区带。 经SDS-PAGE测得该抗菌蛋白的分子量为12,600道尔顿，IEF测得其等电点为9.8。氨基酸组成分析表明，家蝇抗菌蛋白富含脯氨酸，其含量达27.3％。NR／R双向SDS-PAGE证明家蝇抗菌蛋白分子中的两个半胱氨酸残基未形成二硫键。远紫外圆二色谱(CD)分析显示，在生理条件下，家蝇抗菌蛋白的二级结构构象组成为：26.6％α-螺旋，23.7％β-折叠，49.7％β-转角与无规卷曲。在接近细胞膜的疏水环境中，β-折叠含量减少，α-螺旋含量增加。推测家蝇抗菌蛋白的抗菌机理是可能作用于细菌细胞膜，α-螺旋在这一过程中起着重要的作用。 对数种细菌的抗菌活性检测表明，家蝇抗菌蛋白具有较广的抗菌谱，对人病原细菌、昆虫病原细菌及非病原细菌都有抗性，对革兰氏阳性菌的抗性高于革兰氏阴性菌。它不具血细胞凝集活性，亦不能使血细胞发生溶血，具有很高的热稳定性。这种蛋白质不是溶菌酶，而是一种未见报道的抗菌蛋白，根据其各项性质认为它可能属于富含脯氨酸的抗菌蛋白类。 昆虫抗菌蛋白具有传统抗生素不可比拟的优点，有望成为新一代抗菌药物。我们的工作为家蝇免疫机制的进一步深入研究打下了基础，对家蝇抗菌蛋白的潜在应用价值的开发有着重要意义。更多还原

【Abstract】 The immunized haemolymph was produced from Musca domestica(House Fly) larvae by inducement of injuring the body wall with a hypodermic needle. A protein with antibacterial activity was purified at the first time and the properties of the protein had been studied. The protein was obtained by means of boiled water bath, CM-Sepharose ion-exchange chromatography and Sephadex G-50 gel filtration. The purified protein moved as a single band in low pH PAGE. Its molecular weight was 12,600 Dalton by SDS-PAGE and its isoelectric point was 9.8 by IEF. Amino acid composition assay showed that it was rich in proline. NR/R 2-dimensional SDS-PAGE proved that the two cysteine residues in the molecule were not engaged in intramolecular disulfide bridge. The far UV CD analysis indicated that the protein contained 26.6% α-helix, 23.7% β-sheet, 49.7% β-turn and random coil at pH7. In the hydrophobic environments similar to the membrane of bacteria cell, the content of a-helix increased and the content of P-sheet decreased. It was inferred that the helix should be important to the antibacterial activity. The protein had broad antibacterial activity against several human pathogens, insect pathogens and non-pathogens, and the activity against gram-positive bacteria was higher than the activity against gram-negative bacteria. It was not a lectin, nor a lysozyme, but an unknown antibacterial protein with high heat-stability, which should belong to the pro-rich antibacterial protein family.更多还原