黑腹果蝇抗真菌肽Drosomycin同系物的免疫原性和抗真菌活性

Antifungal activity and the immunogenicity of antifungal peptide Drosomycin and its isoforms from Drosophila melanogaster

通过黑腹果蝇Drosophila melanogaster抗真菌肽Drosomycin(Drs)及其同系物Drs-lC和Drs-lE的抗体制备及Western blotting结果,分析了Drs同系物的免疫原性与其抗真菌活性的关系。研究采用了2种技术路线,分别将Drs、Drs-lC和Drs-lE基因构建成与细胞生长因子基因afgf融合的重组表达质粒pET-afgf-Drs、pET-afgf-C和pET-afgf-E,以及通过基因同向串连获得重组表达质粒pRSET-2Drs、-4Drs、-6Drs和pRSET-2E、-4E、-6E,并将这些重组表达质粒转化到BL21(DE3)plysS受体菌进行诱导表达。分离纯化后的融合蛋白afgf-Drs、afgf-C和afgf-E以及串连蛋白4Drs、4Drs-lE分别免疫小白鼠获得相应的抗血清。Western blotting免疫原性检测结果表明,Drs及其同系物与各自的抗血清具有强的免疫反应,同时相互间也有交叉免疫反应,提示它们具有相似的主要抗原决定簇,这些抗原决定簇可能与抗真菌活性无关。同系物之间抗真菌活性的差异可能来源于某些细微结构上的差异。

In order to analyze the antifungal activity and immunogenicity of antifungal peptide Drosomycin （Drs） and its isoforms, Drs-1C and Drs-1E, from Drosophila melanogaster, their antibodies were prepared by two ways. In the first way, Drs, Drs-1C and Drs-1E were respectively fused with afgf gene. In the second way, Drs and Drs-1E were respectively linked into multi-copy genes of Drs and Drs-1E tandem in the same direction. The recombinant plasmids pET-afgf-Drs, pET-afgf-C and pET-afgf-E were constructed through the first way, while pRSET-2Drs, -4Drs, -6Drs and pRSET-2E, -4E, -6E were constructed through the second way. They were then transformed into E. coli BL21（DE3）plysS. After purification, the fused proteins afgfDrs, afgf-C, afgf-E and the concatamer proteins of 4 Drs, 4 Drs-1E were used for preparation of the antiserums by immunizing mice. The results of Western-blot immunogenicity showed that Drs and its isoforms had strong antigen immune reactions with their according antiserums. Cross-reactions between them were also found.These results suggested that they might have the similar main antigen sites. We so inferred that there is no relationship between the main antigen sites and activity sites. The small difference of their structures may be the main mechanism causing the functional divergence of their antifungal activity.