PrGV多角体膜蛋白(PEP)的分析

Analysis of polyhedron envelope protein of Pieris rapae Granulovirus

【目的】对菜粉蝶颗粒体病毒(Pieris rapae Granulovirus,PrGV)多角体膜蛋白(polyhedron envelope protein,PEP)进行研究,为揭示PEP的功能及其在病毒生命过程中的作用提供参考。【方法】以PrGV PEP为对象,通过序列分析、原核表达、抗体制备、Western blotting、胶体金免疫定位、实时荧光定量PCR等方法初步确认该基因定位和表达时相。【结果】PrGV PEP与其他鳞翅目昆虫颗粒体病毒的PEP同源性达到50%左右,但未在核型多角体病毒(Nucleo polyhedro virus,NPV)中发现同源序列。定量PCR分析表明,菜青虫感染PrGV后24h可检测到PEP的存在,随后该基因的表达水平逐渐升高,并在72h达到峰值。通过Western blotting可以在感染PrGV的菜青虫体内检测到大小分别为23ku和15ku的清晰条带,说明制备的多克隆抗体具有较好的特异性。免疫金标记将PEP定位于椭圆型的颗粒体,证明PEP是GV包涵体的重要组成部分。【结论】PrGV PEP在颗粒体病毒中较为保守,可能是PrGV晚期表达的重要基因,参与病毒侵染后期颗粒体结构的形成。

【Objective】This study aimed to investigate the potential roles of polyhedron envelope protein（PEP）in Pieris rapae Granulovirus（PrGV）.【Method】The recombinant GST-fused PrGV PEP was expressed in Escherichia coli and polyclonal antibody against PEP of PrGV was prepared by immunizing a New Zealand rabbit.The location and expression pattern of PrGV PEP were determined using Western blotting,immunoelectron microscopy,and quantitative real-time PCR.【Result】PEP of PrGV showed high homology（about 50%）with PEPs of other Lepidoptera GVs,but had no homologous sequence with nucleo polyhedro virus（NPV）.Quantitative real-time PCR showed that the expression of PrGV PEP was detectable 24 hafter viral infection and peaked at 72 hafter gradually increase.Western blotting analysis verified the specificity of polyclonal antibody against PrGV PEP,with protein bands at 23 ku and 15 ku.The PEP was located in elliptic type particles and was an important component of inclusion body in GV.【Conclusion】The PEP of PrGV was conserved among GVs and participated in the formation of granular structure at the late stage of viral infection.