禽β-防御素6和9基因的克隆、表达及活性分析

Cloning,Expression and Activity Analysis of Avianβ-Defensin 6 and 9

【目的】探究重组禽β-防御素6(AvBD6)、禽β-防御素9(AvBD9)蛋白的原核表达及其生物学特性。【方法】将AvBD6和AvBD9成熟肽基因克隆到表达载体pGEX-6P-1上,进而将重组表达质粒转化到BL21(DE3)pLysS感受态中,用IPTG进行诱导表达后对其进行纯化,SDS-PAGE电泳分析重组蛋白AvBD6和AvBD9的表达情况,继而检测其体外抗菌活性并用透射电镜观察细菌形态变化,检测重组蛋白对鸡红细胞的溶血活性。【结果】重组AvBD6和AvBD9蛋白诱导表达的分子量约为30 ku,且主要在上清中表达。2种重组蛋白对大肠杆菌和金黄色葡萄球菌均具有抗菌活性。电镜观察显示:菌体内容物外泄,损伤严重,并且重组蛋白对鸡红细胞无溶血活性。【结论】本研究制备的AvBD6和AvBD9重组蛋白具有良好的生物安全性和抑菌效果,为进一步研究其抗菌机制及临床初步应用奠定了基础。

[Purpose]To explore the prokaryotic expression and its biological characteristics of recombinant avian defensins 6(AvBD6)and avian defensins 9(AvBD9).[Method]The mature peptide genes of AvBD6 and AvBD9 were cloned into the expression vector pgex-6p-1,and the recombinant expression plasmids pgex-6p-1-AvBD6 and pgex-6p-1-AvBD9 were constructed.Then the recombinant expression plasmid was transformed into BL21(DE3)pLysS.Purification after IPTG induced protein expression SDS-PAGE was used to analyze the protein expression of AvBD6 and AvBD9,then to detect their antibacterial activity in vitro and to observe the morphological changes of bacteria with transmission electron microscope.In addition,the hemolytic activity of recombinant protein on chicken erythrocytes was detected.[Results]By SDS-PAGE analysis,the molecular weight of the induced AvBD6 and AvBD9 is about 30 ku,and it was mainly expressed in the supernatant.The recombinant protein had antibacterial activity against Escherichia coli and Staphylococcus aureus.Electron microscopic observation showed that the bacterial contents leaked out and the damage was serious,and the hemolytic activity of the recombinant protein to chicken red blood cells was very low.[Conclusion]The recombinant protein of AvBD6 and AvBD9 prepared in this study has good biological safety and antibacterial effect,and laid a foundation for further study of its antibacterial mechanism and preliminary clinical application.