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【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(Galleriamellonella)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。
【Aim】 To isolate and purify a protein that inhibits the immune response of Galleriamellonella from the extracellular components of Xenorhabdus bovienii and to study its effect on the pathogenesis of entomopathogenic nematodes and their symbionts Role. 【Method】 The active protein was isolated and purified by ammonium sulfate precipitation and column chromatography, and the active protein was screened by in vivo injection and observation of hemolymph blackening. Fluorescent microspheres and agarose beads were used to evaluate the effect of active protein on blood cells Phagocytosis and coating. The active protein was identified by two-dimensional electrophoresis and mass spectrometry analysis. The gene was cloned by PCR and cloned into prokaryotic expression vector pET 30a. The recombinant protein was purified by affinity chromatography. 【Result】 An insect immunosuppressive protein was purified and named as IDP16. The protein could significantly inhibit the activity of polyphenol oxidase in hemolymph and decrease the phagocytosis and coating of blood cells. The gene was cloned and prokaryotic expressed. The recombinant protein still has immunosuppressive activity. 【Conclusion】 The IDP16 protein produced by the pathogenic bacteria of Berberis inhibits the immune response of insects and plays an important role in the interaction between commensal bacteria and host insects.