甜菜夜蛾核多角体病毒VP39蛋白在苜蓿丫纹夜蛾核多角体病毒中的功能研究

【目的】构建表达甜菜夜蛾核多角体病毒（Spodoptera exigua multiple nucleopolyhedrovirus，SeMNPV） VP39蛋白的vp39假型苜蓿丫纹夜蛾核多角体病毒（Autographa californica MNPV，AcMNPV），明确SeMNPV VP39是否能取代AcMNPV VP39在AcMNPV中行使功能，为深入探究杆状病毒的核衣壳装配机理打下理论基础。【方法】利用Bac-to-Bac杆状病毒表达载体系统在AcMNPV vp39缺失型重组bacmid （bAcvp39KO）的基础上构建携带SeMNPV vp39基因、绿色荧光蛋白基因（Enhanced green fluorescence protein，egfp）和多角体蛋白基因（Polyhedrin，polh）的重组病毒（vAcSevp39:FLAG）。利用Western blotting检测分析SeMNPV vp39在vAcSevp39:FLAG转染的草地贪夜蛾（Spodoptera frugiperda） IPLB-Sf21-AE clonal isolate 9（Sf9）细胞中的表达情况，通过荧光显微镜观察vAcSevp39:FLAG在Sf9中的感染和扩散情况，采用病毒滴度测定并绘制病毒生长曲线检测vAcSevp39:FLAG的感染性芽生型病毒粒子产量，并以电子显微镜观察vAcSevp39:FLAG转染的Sf9细胞中的形态发生情况。【结果】Western blotting检测结果表明，SeMNPV VP39能在v AcSevp39:FLAG转染的Sf9细胞中获得表达；荧光显微镜观察和病毒生长曲线测定结果表明，在vAcSevp39:FLAG转染的Sf9细胞中无感染性的芽生型病毒粒子产生，与AcMNPV vp39缺失型重组病毒（v Acvp39KO）的现象一致；电子显微镜观察发现，与vAcvp39KO不同的是，在vAcSevp39:FLAG转染的细胞中虽然未产生核衣壳，但在感染细胞的核中存在大量空的透明长管状衣壳结构，表明SeMNPV VP39能挽救vAcvp39KO的衣壳结构装配，但在AcMNPV中不具备装配核衣壳的能力。【结论】SeMNPV VP39在AcMNPV中虽能形成衣壳结构，但不能有效装配核衣壳，导致无芽生型病毒粒子和包埋型病毒粒子产生。

Functional analysis of Spodoptera exigua multiple nucleopolyhedrovirus VP39 in Autographa californica MNPV

【Objective】A vp39 pseudotyped Autographa californica multiple nucleopolyhedrovirus(AcMNPV) in which AcMNPV vp39 was replaced with Spodoptera exigua MNPV(SeMNPV) VP39 was constructed to determine whether SeMNPV VP39 could replace AcMNPV VP39 in AcMNPV. This study laid a theoretical foundation for further exploring the nucleocapsid assembly mechanism of baculovirus.【Method】Via Tn7-mediated transposition, the SeMNPV vp39 gene under the control of the AcMNPV vp39 promoter with a FLAG tag prior to the SeMNPV vp39 stop codon, together with the enhanced green fluorescence protein(egfp) and polyhedrin(polh) genes, was inserted into the AcMNPV vp39 knockout bacmid(bAcvp39 KO) polh locus to construct vp39 pseudotyped AcMNPV(vAcSevp39:FLAG). Western blotting was performed to determine whether SeMNPV vp39 was expressed in Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9(Sf9) cells transfected with vAcSevp39:FLAG. The viral replication and infection in Sf9 cells transfected with vAcSevp39:FLAG were monitored using a fluorescence microscope. Viral growth curve analysis was performed by 50%tissue culture infective dose(TCID₅₀) endpoint dilution assay to further evaluate the ability of vAcSevp39:FLAG to produce infectious budded virion(BV). Thin section of vAcSevp39:FLAG-transfected Sf9 cells was observed using an electron microscope to investigate the virus morphogenesis.【Result】Western blotting showed that SeMNPV VP39 was detected in Sf9 cells transfected with vAcSevp39:FLAG. Fluorescence microscopy and viral growth curve analysis showed that no infectious BVs were produced in vAcSevp39:FLAG-transfected Sf9 cells, which was consistent to AcMNPV vp39 knockout recombinant virus(vAcvp39 KO). Electron microscopy demonstrated that, different from vAcvp39 KO, in Sf9 cells transfected with vAcSevp39:FLAG, masses of abnormally elongated empty capsid structures were observed inside the nuclei, but no nucleocapsids formed.【Conclusion】SeMNPV VP39 has the ability to assemble tubular capsid-like structures but not nucleocapsids in AcMNPV, resulting in no BV and occlusion-derived virion produced.