二维电泳和质谱技术鉴定BmNPV包埋型病毒粒子的蛋白组成

Studies on Protein of the Occlusion-Derived Virus of Bombyx mori L. Nucleopolyhedrovirus Using Two-Dimensional Gel Electrophoresis and Mass Spectrometry

【目的】BmNPV包埋型病毒ODV(occlusion-derived virus)粒子在BmNPV感染家蚕初期起了非常重要的作用,主要包含了病毒感染初期所必须的蛋白质,解析其蛋白组成有利于进一步理解病毒和宿主关系,同时也为了进一步了解BmNPV的生活史提供依据。【方法】利用二维电泳对ODV病毒蛋白进行分离,采用考马斯亮兰G-250染色,经质谱鉴定。【结果】共获得70个蛋白点,大部分集中在等电点5～9之间,约占蛋白点总数的61%。其中59个蛋白点适合质谱分析,数据库检索出20种蛋白,其中13种与AcMNPV中鉴定一致,而另7种蛋白是BmNPV中新鉴定出来的。此外,在二维电泳图谱上显示GP41蛋白点有21个,VP39蛋白点有9个。【结论】超速离心结合PCR技术提取ODV蛋白方法可行;二维电泳和质谱联用技术是一种研究病毒蛋白质组快速有效的方法。

[Objective] The occlusion-derived virus （ODV） is produced when nucleocapsids retained in the nucleus are enveloped using virus-induced intranuclear membranes which appear to be derived from the nuclear membrane.It is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. So knowledge of virion composition is a prerequisite for functional investigation. [ Method ] In the present study, two-dimensional gel electrophoresis and mass spectrometry techniques were used to identify the proteins present within or associated with the occlusion-derived virus （ODV） virion, the separated proteins were visualized by Coomassie-Brilliant- Blue-G-250 staining. All protein spots were identified by MS. [Result] The separated proteins revealed 70 spots for the sample. Among them, most proteins were concentrated in pI 5-9, which reached 61% of the total protein spots. The results of this study suggest that 20 proteins were identified with confidence. Interestingly, the result showed that 21 spots of GP41 and 9 spots of VP39 were identified from the gel, respectively. [ Conclusion ] It is feasible to extract ODV protein by a combination of ultracentrifugation and PCR technique; two-dimensional gel electrophoresis combined with mass spectrometry is a rapid and effective way of studying virus proteomics.