凝胶层析法分离家蝇幼虫血淋巴中抗真菌肽的研究

Isolation of antifungal peptides from hemolymph of housefly larvae by Gel filtrationt

目的拟建立家蝇(Musca domestica)幼虫血淋巴中的抗真菌肽的凝胶分离方法。方法取Ⅲ龄家蝇幼虫,以白假丝酵母菌(Candida albicans)作为诱导源,采用针刺法诱导24 h后取血淋巴,加热后分别用凝胶层析和固相萃取分离活性片段,进一步用高效液相色谱法分离纯化,SDS-PAGE比较凝胶层析和固相萃取分离活性组分的区别和分离效果,采用平板滤纸片法检测抗真菌活力。结果免疫血淋巴经凝胶层析得到1个对真菌有抑制作用的组分,其活性比免疫血淋巴经固相萃取得到的1个抗菌组分稳定;SDS-PAGE电泳结果显示,经凝胶层析分离后的活性组分杂蛋白较少,相对分子质量均在14.4×103以下,而经固相萃取的活性组分杂蛋白较多,泳道中有较多大于14.4×103的片段。结论家蝇幼虫经诱导后可产生抗真菌多肽,凝胶层析法能较好地分离该抗真菌肽。

Objective To establish the Gel filtration method of isolating antifungal peptides from hemolymph of housefly larvae（Musca dornestica）. Methods The third instar larvae were stabbed with needle, then put in fungal suspend solution which contains Candida albicans for 24 hours. Isolate antifungal peptides from the hemolymph by heating, Gel filtration, solid phase extraction and HPLC; analyze the antifungal peptides and compare the isolating effect from Gel filtration with that from solid phase extraction by SDS-PAGE electrophoresis. Results The antifungal group isolated by Gel filtration is more steady than the antifungal group isolated by solid phase extraction; SDS-PAGE electrophoresis results shows that the protein molecular weight after Gel filtration is less than 14.4 × 10^3 ; The unnecessary protein of the sample isolated by Gel filtration is less than that by solid phase extraction. Conclusion Housefly larvae can produce antifungal peptides in it＇s hemolymph by induction; The Gel filtration is a good method to isolate the antifungal peptide.