嗜线虫致病杆菌CB6菌株胞内外杀虫蛋白的纯化及比较研究

Purification and Comparison of Toxicity Proteins in Extracellular and Intracellular Products by Xenorhabdus nematophila var. pekingensis

【目的】分离和纯化嗜线虫致病杆菌北京变种(Xenorhabdus nematophila var.pekingensis)CB6菌株胞内和胞外杀虫蛋白,鉴定其蛋白种类。为进一步利用此类杀虫蛋白奠定基础。【方法】采用硫酸铵沉淀、DEAESepharoseFF离子交换柱层析、Butyl SepharoseFF疏水柱层析和SephacrylS-200HR凝胶过滤对该类蛋白进行分离和纯化,采用Native-PAGE和SDS-PAGE技术对所纯化蛋白进行组分分析。【结果】获得达电泳纯的胞内杀虫蛋白E1和胞外杀虫蛋白E2。以2.58μg·ml-1含量E1、以4.21μg·ml-1含量E2喂饲棉铃虫初孵幼虫,对幼虫的生长抑制率分别达62.63%和97.9%。E1、E2经相同纯化参数处理获得的洗脱图相似,经native-PAGE和SDS-PAGE呈现出相似的单带电泳图谱。SDS-PAGE测得E1、E2表观分子量大于212kD,该杀虫蛋白在60℃仍表现出较强的活性。电泳染色结果表明该类蛋白非糖蛋白也非酯蛋白。【结论】CB6菌株胞内杀虫蛋白E1和胞外杀虫蛋白E2可能是同一种(类)蛋白。

[Objective] Toxic proteins in extracellular and intracellular products produced by the strain CB6 of Xenorhabdus nematophila var. pekingensis were purified and compared in order to obtain the basic data of toxic proteins for further study. [Method] Toxic protein fractions from extracellular and intracellular products were purified using the technology of ammonium sulfate precipitation, DEAE-Sepharose FF chromatography, Butyl Sepharose FF chromatography, and Sephacryl S-200 HR chromatography. The protein components were analyzed with the technique of native-polyacrylamide gel electrophoresis （Native-PAGE） and sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）. [Result] Two toxicity protein fractions E1 and E2, respectively, from extracellular and intracellular products were purified to electrophoretic homogeneity. The growth inhibitory percentages against Helicoverpa armigera Htibner larvae was 62.63% and 97.90% with the concentrations of 2.58 μg·ml^-1 E1 and 4.21 μg·ml^-1 E2, respectively. A series of similar liquid chromatograms of E1 and E2 was gained through same purification procedures and parameters. Moreover, they had a similar migration location whether by native-PAGE or by SDS-PAGE. With SDS-PAGE. E1 and E2 have approximate molecular masses higher than 212 kD. They still maintained inhibitory toxicity after heated up to 60℃. Staining experiments showed that the toxicity protein was neither lipoprotein nor glycoprotein. [ Conclusion ] This demonstrated the possibility that the target protein from extracellular section was the same as that from intracellular one.