利用BmNPV多角体包埋外源蛋白的观察

An Observation on Immobilization of Foreign Protein in Polyhedra of BmNPV

家蚕核型多角体病毒在感染晚期大量表达产生多角体蛋白,并形成结构致密的超大分子蛋白质晶体,以有效保护内部包埋的病毒粒子,保证子代病毒的传播。借鉴多角体包埋病毒粒子的原理,观察到多角体也能将外源蛋白质固定入多角体内部。利用能形成多角体的BmNPV Polh+Bac-to-Bac表达系统构建的含有绿色荧光蛋白(EGFP)的重组病毒能在表达、产生正常多角体的同时也表达EGFP蛋白。共聚焦分析表明,该重组病毒感染后形成的多角体能够发射绿色荧光。被包埋的EGFP蛋白放置1个月后仍能检测到绿色荧光,干燥处理30min后仍能检测到荧光。由此说明将多角体与外源基因同时表达,多角体中会包埋外源蛋白,且能较长时间保存外源蛋白的生物活性。这一现象为解决蛋白质长期保存提供了新思路,对于开发含有毒素蛋白的新型生物杀虫剂也有很好的参考意义。

In late phase of BmNPV infection,the virus produces large amount of polyhedrin and forms polyhedra,which contain compact protein crystals.Polyhedron is highly stable,enabling the incorporated virions to persist in unfavorable environment and to have seasonal infections to host larvae.In this study,we investigated whether the foreign protein co-expressed with polyhedrin in recombinant BmNPV could be incorporated into polyhedra like virions.Recombinant baculovirus expressing polyhedrin and EGFP simultaneously was constructed.The results from electron microscopy observation showed that the polyhedra formed from the recombinant baculovirus could incorporate virion as the wild-type BmNPV.Green fluorescence was detected on the surface of purified polyhedra under confocal microscopy.Dissolved BmNPV-EGFP polyhedra emitted green fluorescence after desiccation treatment for 30 min.EGFP bioactivity could also be detected in polyhedra under room temperature after one month.The observation from this experiment showed that the foreign protein,EGFP,could be embedded into polyhedra when co-expressed with polyhedrin and its bioactivity can persist for longer time.It provided a new inspiration for efficient preservation of useful protein and for development of new biopesticides containing toxin proteins.