伯氏致病杆菌IDP16 蛋白抑制大蜡螟的免疫反应

[目的]从伯氏致病杆菌(Xenorhabdus bovienii)胞外组分中分离纯化出能够抑制大蜡螟(Galleria mellonella)免疫反应的一种蛋白,研究其在昆虫病原线虫及其共生菌致病过程中的作用.[方法]采用硫酸铵沉淀和柱层析的方法对活性蛋白进行分离和纯化,通过体内注射并观察血淋巴黑化进行活性蛋白的筛选；采用荧光微球和琼脂糖小球评价活性蛋白对血细胞吞噬、包被作用的影响；采用双向电泳结合质谱分析对活性蛋白进行鉴定,设计引物用PCR的方法克隆其编码基因,利用pET 30a载体进行原核表达,以亲和层析纯化重组蛋白.[结果]纯化得到一个昆虫免疫抑制蛋白,命名为IDP16,该蛋白可显著抑制大蜡螟血淋巴中的多酚氧化酶活性,降低血细胞的吞噬和包被作用.克隆得到其编码基因并进行了原核表达,重组蛋白仍具有免疫抑制活性.[结论]伯氏致病杆菌产生的IDP16蛋白能够抑制昆虫的免疫反应,在共生菌和宿主昆虫互作过程中起着重要的作用.

An IDP16 protein from Xenorhabdus bovienii depresses the immune response in Galleria mellonella

[Objective] A protein with insect immunodepressive activity was purified from Xenorhabdus bovienii,a bacterial symbiosis of entomopathogenic nematode.To determine the function of this protein in the pathogenesis of bacterium-nematode symbiosis,we detected the effect of this protein on immune response in Galleria mellonella.[Methods] Proteins from extracellular extact of X.bovienii were purified using ammonium sulfate precipitation,HiTrap Q HP chromatography,HiTrap Butyl FF chromatography,and HiTrap SP HP chromatography.Intra-hemocoel injection assay followed by observation of hemolymph melanization was used for activity determination.Fluorescent microspheres and sepharose beads were used to assess the effect of purified protein on phagocytosis and encapsulation of hemocytes,respectively.The purified protein was identified by 2-D and MS anlysis.The full-length encoding gene was cloned by PCR,and expressed with pET 30a in Escherichia coli.The recombinant protein was purified by Ni-NTA affinity chromatography.[Results] An immunodepressive protein was purified and designed as IDP16,which can inhibit the phenoloxidase activity,and weaken the phagocytosis and encapsulation of Galleria mellonella hemocytes.The encoding gene was then cloned and expressed in E.coli.The recombinant protein was also determined to be immunodepressive.[Conclusion] The IDP16 protein from Xenorhabdus bovienii can depress the immune response in insect,which may play an important role in bacteria-host interaction.