禽防御素在雏鸡抗鸡白痢沙门菌感染中的表达及其分子设计

Avian beta-defensins' expression in chickens infected with Salmonella Pullorum and molecular designing for reshaped defensin

为了探寻有效防治雏鸡感染鸡白痢沙门菌的新型防御素生物制剂,建立雏鸡感染鸡白痢沙门菌（S.Pullorum）的模型,感染3～24 h后检测十二指肠组织禽防御素（AvBDs）抗感染的防御性表达水平,分析AvBDs的理论等电点,选择活性强且结构稳定的AvBD,对其进行分子设计,以期构建出理论生物活性更强且结构更稳定的改型防御素.结果表明,与对照组相比,雏鸡感染鸡白痢沙门菌3～24 h后十二指肠AvBD-6的表达水平明显上升,差异显著（P＜0.05）,结合理论等电点分析,选择AvBD-6做设计出发肽,用天冬氨酸替换AvBD-6原有的中性氨基酸,并在C端增加抗菌肽BF2衍生物的α螺旋,成功构建了改型防御素AvBD6-BF2,其理论生物活性与结构稳定性均强于AvBD6出发肽.本研究将为AvBDs生物制剂用于防治鸡白痢沙门菌病的可行性提供参考.

In order to develop an alternative antibiotic for the treatment of avian Salmonellosis, we established S.pullorum infection model with 14-day old chickens. We detected tile expression profile of avian beta defensins （AvBDs）in chickens at different time points by RT-PCR assay and analyzed the theoretical isoeleetric point of them. The AvBD-6, which was most highly expressed and stable, was designed as a new reshaped defensin based on its physicochemical characteristics, secondary structure, transmembrane characteristics, molecular model and DNA/RNA binding experiment. The resuhs showed that the expression levels of AvBD2, 4, 5, 6, 7 and 9 in experimentally infected chickens were significantly increased （P〈0.05）for 3-24 h. After 24 h, the expression levels of AvBD 1,2,6,7, 11 and 12 were higher than that of other AvBDs. So AvBD6 may be the advantageous β-defensin based on the results on theoretical isoelectric points of AvBDs. It had the common nature of antimicrobial peptides-cationic and amphiphilic and was likely to target the bacterial cytoplasm. AvBD6 had no α-helix. So we designed the AvBD6-BF2 reshaped defensin with three strands and an α-helical structure, which increased alpha helix BF2 derivatives, strengthened the membrane action ability and reduced the isoeleetric point.