家蝇幼虫血淋巴抗真菌肽MAF-1的分子特性分析及其抗真菌作用

Characterization of the antifungal peptide MAF-1 from the hemolymph of Musca domestica larvae and its antifungal activity

制备家蝇Musca domestica幼虫血淋巴中的抗真菌肽,对其分子特性及抗真菌机制进行研究。通过C18柱固相萃取、反相高效液相色谱相结合的方法,成功制备到家蝇幼虫血淋巴抗真菌肽MAF-1。SDS-PAGE分析结果表明,MAF-1的分子量为17.136kDa。通过圆二色谱测出水溶液中MAF-1主要以α螺旋及β折叠为主。扫描电镜观察结果显示,MAF-1作用白假丝酵母菌Candida albicans1h后,部分真菌出现表面凸凹不平,细胞结构模糊;随着作用时间的延长,表面形成很明显的凹陷,皱缩,个别菌体破裂,内容物外泄,病变真菌发生率高于对照组。单细胞凝胶电泳(single-cell gel electrophoresis,SCGE)结果显示,正常对照组的真菌呈现典型的圆形,而实验组则出现明显的拖尾现象即形成"彗星"样细胞特征,细胞迁移距离明显高于对照组。SDS-PAGE图谱显示,MAF-1作用后的白假丝酵母菌菌体蛋白谱带一些条带明显变浅,条带数下降,甚至消失。结果提示MAF-1可能具有独特的抗真菌机制。

To study the antifungal mechanism of Musca domestica antifungal peptide, MAF-1 was successfully prepared by solid phase extraction （SPE） and RP-HPLC from the hemolymph of M. domestica larvae. MAF-1 exists mainly as a-helices and g-sheets in aqueous solution, as revealed by analysis of circular dichroism spectra. The results detected by scanning electron microscopy showed that the membranes of some Candida albicans fungi were depressed, shrinked and ruptured at 1 h after treatment with MAF-I. The results of single-cell gel electrophoresis showed that the ＂comet＂-like cells were observed in the experimental group; however, typical round fungi were found in the normal control group. SDS-PAGE analysis showed that some protein bands were significantly weaker, the number of bands decreased and some bands even disappeared when C. albicans was treated with MAF-1. The results suggest that MAF-1 may have a unique antifungal mechanism.