家蝇幼虫淋巴液的抑菌作用及其SDS-PAGE分析

SDS-PAGE analysis of the antibacterial activity of the hemolymph from housefly larva

目的观察针刺诱导家蝇幼虫血淋巴抗菌物质抑菌作用与抗菌分子。方法室内饲养的家蝇三龄幼虫,经针刺体壁处理后48h,提取其血淋巴,观察单独使用血淋巴和血淋巴与氨苄青霉素混合对9种应试细菌的抑菌效果,血淋巴与抗真菌药氟康唑混合对酵母真菌抑菌效果,应用SDS-PAGE分析血淋巴抗菌分子表达。结果针刺家蝇幼虫后48h的血淋巴,作用于大肠杆菌、金黄色葡萄球菌、白色葡萄球菌、福氏痢疾杆菌、变形杆菌、枯草杆菌、伤寒杆菌、副伤寒杆菌、黄色小球菌,结果显示:针刺家蝇幼虫后48h的血淋巴对试验9个标准株细菌,均产生明显抑菌环,经单因素方差分析,显示各抑菌试验组的抑菌效果,皆有显著差异(P<0.001)。其中对黄色小球菌抑菌活力最强。1μ1氨苄青酶素与7μ1针刺诱导家蝇幼虫淋巴液混合对白色葡萄球菌抑菌效果呈现最好的加强抑菌作用。针刺诱导家蝇幼虫血淋巴与抗真菌药物氟康唑混合对酵母真菌抑菌效果呈现明显协同增强作用。SDS-PAGE分析结果显示:针刺幼虫后发育的成蝇和针刺幼虫血淋巴的电泳条带与未针刺者相比,针刺幼虫后发育的成蝇,在24000￣97000范围内表达尤为增强,在12000附近有一条特异性条带。针刺家蝇3龄幼虫血淋巴SDS-PAGE分析显示,在24000、35000、40000￣96000范围内表达明显增强,其中35000、12000条带较为特异。结论本研究进一步验证了家蝇血淋巴的抑菌活性,发现针刺诱导家蝇幼虫血淋巴对黄色小球菌抑菌活力最强,氨苄青霉素与家蝇幼虫淋巴液混合对白色葡萄球菌抑菌效果呈现最好的加强抑菌作用,针刺诱导家蝇幼虫血淋巴与抗真菌药物氟康唑混合对酵母真菌抑菌效果呈现明显增强作用,上述发现在国内外报道较少,本研究结果,为进一步开发这类抗菌物质,提供了实验资料。

Objective To study the inducible antibacterial activity of the hemolymph from housefly larva and analyze the antibacterial molecules. Methods The hemolymph was collected from the third instar housefly larvae 48 h after pricking treatment. Nine standard bacterial strains were used for determination of the antibaterial activity of the collected hemolymph and its combination with ampicillin. The anti-yeast activity of the hemolymph and its mixture with fluconazol was also assayed. The antibacterial molecules in the hemolymph was analyzed by SDS-PAGE. Results The growth of E.coli, Staphylococcus aureus, Staphylococcus albus, subserotypes of Shigeila flexneri, Bacillus proteus, Bacillus subtilis, Bacillus typhi, Bacillus paratyphosus, and Micracoccus lysodeikticus could be inhibited by the hemolymph collected from housefly larva, and the effect differed significantly between the groups （P〈0.001）. The hemolymph produced the strongest antibacterial activity against Micrbcoccus lysodeikticus, and the combination of the hemolymph with ampicillin most conspicuously inhibited the growth of Staphylocaccus albus. The hernolyph and fluconazol exhibited obvious synergistic effect against yeast. SDS-PAGE identified some specific antibacterial molecules in the hemolymph. Conclusion The induced hemolymph from housefly larva possesses strong antibacterial and antifungal activities especially against Microcaccds lysodeikticus. The hemolymph in combination with ampicillin produces the strongest effect against Staphylococcus albus, and fluconazol can significantly enhance the anti-yeast activity of the hemolymph through a synergistic mechanism.