杆状病毒介绍
昆虫表达系统介绍
杆状病毒是已知昆虫病毒中最大的类群发现最早、研究最多且实用意义也是最大的昆虫病毒。
杆状病毒表达载体系统Baculovirus expression vector systemBEVS自1983年问世以来Smith Summer1983Maeda et al 1984由于其具有高效的表达能力、安全易操作等特点已成为研究和生产各种原核、真核蛋白的用力而普及的工具已成功表达了大量的外源基因几乎覆盖了所有物种可开发利用的大部分基因。
1 分类杆状病毒——有囊膜的双股DNA大型病毒仅限于无脊椎动物有尾噬菌体目Caudovirales 杆状病毒科Baculoviridae 核型多角体病毒属NucleopolyhedrovirusNPV ———苜蓿蠖核型多角体病毒Autographa Californica NPV ———家蚕核型多角体病毒Bombyx mori MPV 颗粒体病毒属GranulovirusGV ———苹果蠹蛾颗粒体病毒Cydia pomonella GV 多角体与颗粒体的区别NPV和GV均有包涵体形式NPV的称多角体后者称为颗粒体多角体内含有许多病毒子都在宿主细胞核内存在颗粒体内含一个病毒子偶尔是两个绝大部分在核内形成但某些GV在胞浆中形成颗粒体2 发育循环杆状病毒包含两个独特的时相又称双相生活循环biphase life cycle 第一相出现在PI0-24hr 核衣壳于胞核内病毒发生基质上进行装配通过细胞质出芽获得囊膜这种病毒称为细胞释放型病毒子Cell-released virusCRV又称胞外型病毒Extracellular virusECV或出芽型病毒Budded virusBV 第二相出现在PI20-72hr 当20hr后核内出现病毒包涵体时第二相显著出现一直持续到感染细胞溶解为止72hr 随着第二相开始CRV急剧减少从第二相开始留在核内的核衣壳被封入核内新装配的囊膜中以后许多核内获得囊膜的病毒子被包埋入多角体蛋白的结晶基质中逐渐形成多角体。
杆状病毒1
杆状病毒的研究进展1．杆状病毒作为基因治疗载体的研究进展：将基因高效导入细胞是基因治疗的技术关键。
我们构建了一个含有CMV启动子启动的绿色荧光蛋白基因的重组AcMNPV，并用其作为基因转移载体转导哺乳动物原代椎间盘细胞。
AcMNPV介导报告基因－绿色荧光蛋白基因在该原代细胞中能高效表达，200 MOI时病毒在该细胞中表达报告基因百分比最高（87%），且表达时相可持续较长时间，此外，杆状病毒的转导对细胞没有明显毒性。
体内实验表明，杆状病毒能有效、无毒地转导兔椎间盘细胞。
另外，通过比较杆状病毒介导的外源性基因在正常髓核细胞与退变髓核细胞中的表达，发现两者之间无明显差异，均能高水平表达。
因此，杆状病毒转染椎间盘髓核细胞的效率与椎间盘退变程度无关。
由此可见，杆状病毒在作为椎间盘疾病的基因治疗转移载体方面，具有巨大的潜力。
进一步通过体内外实验还发现，杆状病毒能进入人椎间盘细胞中并介导外源基因的高效表达，尤其是Sox9基因的表达，对于治疗椎间盘退化具有明显效果。
进一步证实了杆状病毒作为高效、安全的基因治疗载体的应用前景，该研究成果发表于国际医学权威杂志SPINE上（Liu et al., 2006）。
同时，我们发现杆状病毒能刺激哺乳动物细胞产生非特异的免疫反应，并对其机制进行了研究（Liang et al., 2006）；分析了杆状病毒转导家禽细胞以及组织的特性和转导机制（Song et al., 2006）；并且，我们还发展了一类新型的基于杆状病毒F蛋白的真核表面展示系统，为具有自主知识产权的靶向性基因治疗载体的构建进行了有益的探索（Mao et al., 2006）。
2．杆状病毒为载体的基因工程疫苗的研究：我所对乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）的杆状病毒疫苗进行了初步研究：利用细菌和昆虫病毒表达系统成功地表达了HCV的E2和NS2蛋白，并分析了其免疫学特性（刘超红等，2006）；构建了插入1.3倍HBV全基因组的重组杆状病毒，在哺乳动物细胞中获得了HBVS抗原和E抗原的表达，并产生病毒颗粒（于德敏等，2006）；利用杆状病毒构建了SARS CoV 类病毒颗粒（VLP）并对其免疫学特性进行了详细的研究，以期为SARS CoV疫苗研究提供一个安全、有效的方法，并为SARS CoV组装的基础研究提供技术平台（Lu et al., submitted）；同时，我所还与华中科技大学同济医学院积极合作，开展了血吸虫杆状病毒疫苗的研究（Yu et al., 2006），发现以杆状病毒载体为基础的基因工程疫苗与蛋白疫苗联合应用获得的免疫效果和保护效果最佳，为血吸虫的免疫接种提供了新的途径及策略，具有良好的开发应用价值。
杆状病毒——昆虫细胞表达系统
实验材料：1. 重组杆状病毒质粒：Bacmid/nsp-6及阳性对照Bacmid/CAT，已构建成功。
2. 昆虫细胞Sf9、High Five及其相关培养基、转染试剂均购自Invitrogen公司。
抗His单克隆抗体购自Oncogene公司，CAT-ELISA试剂盒购自Roche。
实验步骤：一、昆虫细胞转染：1． Sf9细胞计数，取6孔板中的两孔，每孔加入9×10 5个细胞(其中一孔设为正常对照)，并以全培培养至少1小时，使细胞贴壁。
2．准备重组质粒和细胞转染试剂的混合物：a. 溶解1μg纯化重组杆状病毒重组质粒于100μl 无添加成分的Grace’s Medium。
b. 转染试剂充分摇匀后取6μl加入100μl 无添加成分的Grace’s Medium，混匀。
c. 将上述稀释好的质粒及稀释好的转染剂混匀，室温孵育20min。
3．重组质粒与转染剂混合液孵育的同时，以2ml无添加成分的Grace’s Medium洗涤待转染的一孔细胞并弃去洗液。
4．取0.8ml无添加成分的Grace’s Medium加入质粒与转染剂的混合液中，轻轻混匀后，总体积约为1ml。
加入上步洗涤后的细胞孔中，27℃继续培养5h。
5．移除质粒、转染剂混合物，加入2ml全培。
27℃湿盒孵育，直到病变现象产生。
二、病毒贮液的制备：1. 病毒感染晚期（正常24-72h）可见细胞停止生长、黏附，呈颗粒状外观。
即收集含病毒的培养上清，500g离心5min，去除细胞和碎片。
2. 上清即为P1病毒贮液，移入新的离心管中4℃避光保存。
长期保存分装冻存于-80℃。
3. 病毒贮液的扩增，按以下公式进行所需病毒P1贮液的量：感染所需病毒贮液量(ml)=[MOI(pfu/cell) ×细胞数÷病毒贮液效价(pfu/ml)]注：若不进行病毒空斑测定，P1贮液效价按照1×10 6到1×10 7计。
4. 扩增P1液制备P2病毒贮液方法如下：a. 转染当天，取2×106个细胞/孔加入六孔板中，贴壁生长至少1h。
关于生物防治的论文
昆虫杆状病毒的研究与应用现状摘要：杆状病毒是节肢动物的专性病原物，多见于昆虫纲的鳞翅目昆虫。
在昆虫杆状病毒中昆虫杆状病毒表达载体系统的建立和发展，被誉为20世纪80年代真核表达研究领域的一个重大进展。
文章重点介绍了昆虫杆状病毒表达载体系统的研究和在基础研究领域，农、林业的应用现状。
关键词：昆虫杆状病毒表达载体系统，基础研究，农、林业昆虫杆状病毒包含核型多角体病毒和颗粒体病毒两大类，是昆虫专一性病原物，对目标害虫致病性强，不产生抗药性，田间释放安全、环保。
同时由于病毒粒子被抗逆性很强的蛋白（多角体蛋白或颗粒体蛋白）所包裹，在环境中较为稳定，制成农药制剂后，货架寿命相对较长，使用方便，具有很强的商品属性。
其中，昆虫杆状病毒表达载体系统具有表达水平高、表达产物可进行翻译后加工，并可通过感染昆虫幼虫而实现大规模低成本生产基因工程产品。
早在上世纪70年代，昆虫杆状病毒就被美国食品与药品管理局和世界卫生组织推荐为安全的生物杀虫剂用于害虫的防治。
当前，环境保护和食品安全问题日益受到关注，如何解决植物保护和环境污染、农药残留之间的矛盾，是植保工作者必须面对的课题。
昆虫杆状病毒杀虫剂作为生物农药中的重要成员，应该为此做出贡献。
我国昆虫病毒杀虫剂的产业化开发已有30年的历史，有过骄人的成绩，然而同其它产业化成功的生物农药相比（如Bt、井冈霉素、阿维菌素等），无论在生产规模、质量标准、市场份额、社会影响等诸多方面，都存在巨大的差距。
一、昆虫杆状病毒的研究昆虫杆状病毒是最大的环状单一双链DNA病毒，其基因组在90～230 kb之间，具有编码上百种蛋白质的能力。
该病毒基因组可在昆虫细胞核内进行复制和转录，其巨大的DNA复制后组装在杆状的核衣壳内。
由于昆虫杆状病毒DNA具有大量的非复制必需区，能容许基因缺失或替换，且其较大的柔软性，能容纳大片段外源DNA的插入，这为昆虫杆状病毒的重组提供了广阔发展空间。
在昆虫杆状病毒的研究中，杆状病毒表达载体系统是一个以昆虫杆状病毒为外源基因载体，以昆虫和昆虫细胞为受体的表达系统。
对虾杆状病毒病
组织损伤
03
病毒导致对虾的肝胰腺、鳃丝等组织损伤，影响其正常生理功
能。
对虾品种的敏感性差异
敏感性差异
不同品种的对虾对杆状病毒的敏感性存在差异 。
高风险品种
部分品种的对虾容易感染杆状病毒，如白对虾 、斑节对虾等。
低风险品种
部分品种的对虾对杆状病毒相对不敏感，如草虾、沼泽对虾等。
04
病毒的检测和诊断
病毒的分类和特征
分类
SBV属于杆状病毒科，是虾类的专性病毒。
特征
SBV的主要特征是其杆状形态，长度约为750纳米，直径约为110纳米。该病毒 具有囊膜，内含有一个大的、线性的、dsDNA基因组。
病毒的基因组和蛋白质
基因组
SBV的基因组为dsDNA，大小约为80kb，编码约20个蛋白 质。基因组被分成两个主要部分，即大端（L）和小端（S） ，它们分别编码病毒的复制和包装相关蛋白。
针对对虾杆状病毒病的威胁，提出了多种可持续发展 的策略，包括改变养殖模式、优化养殖环境、疫苗研 发和应用等，以保障对虾养殖业的可持续发展。
THANK S感谢观看
日期:
对虾杆状病毒病
汇报人：
目录
• 病毒概述 • 病毒的流行病学 • 病毒的致病性和症状 • 病毒的检测和诊断 • 病毒的预防和控制 • 研究进展和未来趋势
01
病毒概述
病毒的发现和命名
发现
对虾杆状病毒病是在1998年首次发现于中国对虾上，后来在其他对虾种类上也 发现了该病毒的存在。
命名
该病毒被命名为对虾杆状病毒（Shrimp Banded Virus，SBV），属于杆状病 毒科。
注意药物的使用方法和剂量，确保药 物的安全和有效性，避免药物残留和 对对虾的影响。
杆状病毒基因转导的技术和应用
杆状病毒基因转导的技术和应用随着科学技术的不断进步，人类对基因的认识和应用也逐渐深入。
在基因工程领域，杆状病毒基因转导技术是一种十分有前途的技术。
本文将介绍杆状病毒的基本情况、杆状病毒基因转导技术的原理和应用。
一、杆状病毒的基本情况杆状病毒是一种单链RNA病毒，是植物病毒中最重要的一种。
它可以侵入植物细胞内部，通过自身的基因组合成一种非常具有活力的蛋白质，然后传递给其他细胞。
由于其生物学特性，许多疾病和社会问题一直没有得到很好的解决。
因此，了解杆状病毒的基本情况是基因转导技术研究的重要前提。
二、杆状病毒基因转导技术的原理杆状病毒基因转导技术是将外源DNA引入植物细胞中的一种方法。
一般来说，它是通过将带有目的基因的载体构建成病毒载体，然后通过感染植物细胞来实现目标基因的转录和翻译的。
在这个过程中，载体的构建是很关键的。
一般来说，载体需要包括一个病毒颗粒功能区、一个基因组合成的目标DNA功能区和一个表达基因的启动子区。
通过这三个功能区的合成，我们就可以得到病毒颗粒，进而实现基因转导。
三、杆状病毒基因转导技术的应用杆状病毒基因转导技术的应用非常广泛，在基因工程、生物学研究和生产过程中都有非常重要的作用。
以下是几个常见的应用领域：1. 植物基因研究杆状病毒基因转导技术可以用于植物基因的研究和功能分析。
通过这种方法，可以快速有效地将外源基因引入植物细胞，研究它们的表达和功能等方面的问题。
这能够提高我们的基因研究的精度和效率，也可以为我们了解植物的种类和特性提供有用的信息。
2. 植物基因工程杆状病毒基因转导技术还可以用于植物的基因工程领域。
通过这种方法，我们可以有效地转化作物和一些经济植物，从而对其进行基因编辑和优化。
这有助于我们提高作物产量、提高经济效益、改善植物的品质，为人们生活和经济发展做出贡献。
3. 疫苗生产杆状病毒基因转导技术也可以用于疫苗的生产。
一些病原体所带有的代谢基因、毒力基因等，可以被杆状病毒转导载体所调控，然后被表达出来。
杆状病毒资料
DOCS
DOCS SMART CREATE
杆状病毒：基本概念与深入研究
01
杆状病毒的基本概念及其特征
杆状病毒的分类与分布
杆状病毒属于病毒科
• 包含多个属，如昆虫杆状病毒属、哺乳动物杆状病毒属等 • 病毒分布广泛，包括昆虫、哺乳动物、鸟类等
昆虫杆状病毒属
• 主要感染昆虫，如果蝇、蚊子、蝴蝶等 • 具有宿主特异性，不同病毒针对不同的昆虫宿主
杆状病毒与宿主免疫系统具有相互影响的关系
• 病毒可通过抑制宿主免疫反应，逃避宿主免疫清除 • 宿主免疫系统可通过识别和清除病毒，抑制病毒复制和扩散
04
杆状病毒的应用与研究进展
杆状病毒在基因治疗中的应用
• 杆状病毒可作为基因载体，将目的基因导入宿主细胞 • 病毒具有宿主范围广、基因容量大等优点，适用于多种基因治 疗策略 • 通过基因编辑技术，改造病毒载体，提高基因导入效率和安全 性
哺乳动物杆状病毒属
• 主要感染哺乳动物，如猴子、松鼠、蝙蝠等 • 部分病毒可引起人类疾病，如出血热、脑炎等
杆状病毒的形态与结 构
• 杆状病毒呈长棒状形态，直径约20-30纳米，长度可达几百纳 米
• 病毒核衣壳由核壳蛋白组成，具有螺旋对称结构 • 病毒基因组为单链DNA或双链RNA，线性或环状 • 病毒基因组两端具有反向重复序列（ITR），有助于病毒复制和 组装 • 杆状病毒具有包膜结构，包膜上含有病毒蛋白，有助于病毒感染 宿主细胞
杆状病毒通过吸附、内吞和解包等过程感染宿主细胞
• 病毒吸附在宿主细胞表面，通过受体结合进入细胞 • 病毒通过内吞作用进入细胞，形成内质网或溶酶体内的病毒颗粒 • 病毒在细胞内解包，释放基因组，开始病毒复制和表达
杆状病毒
Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74is prerequisite in per os infectionJeffrey M.Slack,1Susan wrence,2Peter J.Krell3and Basil M.Arif1Correspondence Jeffrey M.Slack jslack@nrcan.gc.ca 1Great Lakes Forestry Centre,Sault Ste Marie,ON P6A2E5,Canada2US Department of Agriculture,Beltsville,MD20705-2350,USA3Department of Molecular and Cellular Biology,University of Guelph,ON N1G2W1,CanadaReceived28March2008 Accepted25June2008Baculovirus occlusion-derived virions(ODVs)contain a number of infectivity factors essential for the initiation of infection in larval midgut cells.Deletion of any of these factors neutralizes infectivity by the per os route.We have observed that P74of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor(SBTI).Presently,biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity.We developed a method to rescue per os infectivity of a P74null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74.We used this plasmid rescue method to functionally characterize P74.A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function.Mutagenesis of R195,R196and R199compromised per os infectivity and rendered P74resistant to midgut trypsin.INTRODUCTIONBaculoviruses are a group of arthropod-specific viruses (Zanotto et al.,1993)that have been applied as insecticides and as vectors for the expression of exogenous genes.They have a biphasic replication strategy producing two distinct viral phenotypes:the budded virion(BV)and the occlusion-derived virion(ODV).Both virion phenotypes have bilayer lipid envelopes surrounding bacillus-shaped nucleocapsids and contain double-stranded,circular DNA genomes.However,the integral protein composition of their envelopes and their roles in infection are distinct. BVs spread viral infection throughout host tissues by attaching to and entering host cells via receptor-mediated endocytosis(Volkman&Goldsmith,1985).Endosomal acidification triggers envelope fusion with the endosomal membrane to release the viral nucleocapsid into the host cell(Leikina et al.,1992).In the case of group I alphabaculoviruses(Jehle et al.,2006),budding,attach-ment and envelope fusion are mediated by the viral protein GP64(Blissard&Wenz,1992)and for other baculovirus types these processes are mediated by an F protein (Pearson et al.,2000;Westenberg et al.,2002).ODVs are required in the horizontal transmission of baculoviruses between insect hosts(Kozlov et al.,1986). The ODVs of all baculoviruses are occluded into proteinaceous occlusion bodies(OBs)prior to release from the host(Rohrmann,1986).The distinctive protein structure of OBs has a dual function.It serves to protect virions from deleterious environmental factors,and also acts as a delivery mechanism to transport the ODVs to the alkaline midgut where the cells are susceptible to infection. ODV envelopes are derived from the inner nuclear membrane(Braunagel&Summers,1994)and contain envelope proteins that can survive the protease-rich environment of the insect midgut.ODVs attach to midgut columnar epithelial cells and fuse their envelopes directly with the cell membrane(Kawanishi et al.,1972;Tanada et al.,1975;Granados,1978;Horton&Burand,1993).A number of ODV envelope proteins are essential per os infectivity factors(PIFs),including P74(Kuzio et al., 1989),PIF1(Kikhno et al.,2002),PIF2(Pijlman et al., 2003)and PIF3(Ohkawa et al.,2005).PIF1,PIF2and P74 have been implicated to be involved in ODV attachment to midgut cells(Haas-Stapleton et al.,2004;Yao et al.,2004; Ohkawa et al.,2005).P74,the first identified PIF(Kuzio et al.,1989),is N-terminally exposed on the ODV surface and C-terminally anchored in the ODV envelope(Faulkner et al.,1997; Rashidan et al.,2005).These investigations are thePublished online ahead of print on23July2008as DOI10.1099/vir.0.2008.002543-0.Supplementary tables are available with the online version of this paper.Journal of General Virology(2008),89,2388–2397DOI10.1099/vir.0.2008/002543-0 23882008/002543Printed in Great Britaincontinuation of previous ones that showed the N terminus of P74to be specifically cleaved by insect midgut trypsins (Slack&Lawrence,2005).In the present study we found that Trichoplusia ni larvae were more susceptible to per os infection by Autographa californica multiple nucleopolyhe-drovirus(AcMNPV)when trypsin was added to the diet and they were less susceptible in the presence of soybean trypsin inhibitor(SBTI).We have developed a transient plasmid-based assay to evaluate the ability of P74mutants to rescue per os infectivity of a P74null virus.It has been established that a P74fused with the green fluorescent protein can function in place of native P74(Yao et al.,2004).In this assay,insect cells were co-transfected with P74null virus DNA and a plasmid expressing the p74gene fused in-frame with the enhanced green fluorescent protein(egfp)gene. Earlier,we proposed that P74is cleaved at arginine residue R156(Slack&Lawrence,2005)and this study examines the functional effects on P74of mutating R156and nearby trypsin cleavage site residues.We co-transfected insect cells with mutant p74–EGFP-expressing plasmids and P74D virus DNA.OBs produced in these cells were harvested and fed to insects.It was clearly shown that R156is not required for P74function and that instead a nearby cluster of trypsin cleavage sites at residues R195,R196and R199 were important for function.Experiments with insect brush border membrane vesicles(BBMV)confirmed that absence of these trypsin cleavage sites eliminated the specific cleavage of P74.METHODSCell lines and viruses.Spodoptera frugiperda Sf9cell lines were propagated in10%v/v fetal bovine serum,2.5%w/v tryptose broth-supplemented Grace’s(FBS/Grace’s)media(Sigma-Aldrich).The parental AcMNPV virus used in this study was isolate HR3(Brown et al.,1979).The P74null(P74D)AcMNPV virus used in this study was AcLP4(Faulkner et al.,1997).This AcLP4virus has the b-galactosidase reporter gene inserted into the middle of the native p74 ORF of AcMNPV and it produces the N-terminal194aa of P74fused in-frame with b-galactosidase.Plasmid constructs.The plasmids,pBAC-5-EGFP and pBAC-5-p74-EGFP,were previously described as pBAC-5-GFP and pBAC-5-p74-GFP(Slack et al.,2001).pBAC-5-p74-EGFP contains the AcMNPV p74ORF fused at the39end in-frame with the jellyfish EGFP ORF(Zhang et al.,1996).pBAC-5-p74-EGFP is based on the pBAC-5baculovirus transfer plasmid(Novagen).Site-directed mutagenesis.All mutants were made using muta-genic PCR primers and the Deep Vent polymerase(New England Biolabs).PCR amplifications were done with35cycles of95u C 1min,45u C1min30s and72u C1min45s.The seven P74mutants,R114Q,R119Q,R132Q,K138Q,R156Q, RK184/186QQ and RRR195/196/199QQQ,were generated by amp-lifying two PCR products that,when ligated together,yield an823bp fragment of the59end of the p74ORF.This823bp fragment contained Nco I and Bst XI sites that permitted cloning in place of the corresponding p74ORF region in pBAC-5-p74-EGFP.The first or‘left’PCR products were made with a common Nco I site (underlined)-containing primer,p74M1NcoI-59(59-AAGCACCA-TGGCGGTTTTAACAGCCGT-39)and phosphorylated mutagenic primers.The second or‘right’PCR products were made with a common Bst XI site(underlined)-containing primer,p74D274BstXI-39(59-AAGCACCATGGCGGTTTTAACAGCCGT-39)and primers that corresponded to the positions immediately on the59end of the mutagenic primers(see Supplementary Table S1,available with the online version of this paper).Left and right PCR products were fractionated by agarose gel electrophoresis and purified using Qiaex II glass milk(Qiagen).The corresponding left and right PCR products were ligated using T4 DNA ligase and the ligation was amplified by PCR using the primers p74M1NcoI-5and p74D274BstXI.The pBAC-5-p74-EGFP plasmid contained two Bst XI sites and was partially digested with Bst XI prior to Nco I digestion.The resulting vector minus the original p74Nco I/ Bst XI fragment was fractionated by agarose gel electrophoresis and purified by glass milk.The PCR product was cut with Nco I and Bst XI and ligated into the corresponding sites in the p74ORF.The P74mutants R195Q,R196Q,R199Q,R195Q/R196Q,R196Q/ R199Q and R195Q/R199Q were made within a395bp Eco RI/Sac II fragment of the p74ORF.All of these mutagenic PCR products were amplified with the Sac II-containing primer p74R319SacII-39(59-CGTTACCGCGGTAATTGTATGCGATC-39)and an Eco RI site containing mutagenic primer(see Supplementary Table S1).PCR products were fractionated by agarose gel electrophoresis and purified using glass milk.The pBAC-5-p74-EGFP plasmid and PCR products were cut with Eco RI and Sac II,fractionated by agarose gel electrophoresis,glass milk-purified and ligated together.All p74mutants were sequenced(University of Guelph Molecular Supercentre)in both directions using primers p74Sequencing1LP36 (59-AAACCCAAGTTCAGTCTGC-39)and p74Sequencing2RP37(59-AAGTGACAAAGATCGTGTC-39).OB preparation.OBs were obtained from infected Sf9insect cells. Infected cells were suspended in media and then centrifuged for 5min at1000g.Pellets were suspended in PBS(120mM NaCl, 10mM Na2HPO4,2.5mM KCl,pH6.2)and the OBs were released with SDS(0.3%w/v)and centrifuged for10min at2000g.OB pellets were washed several times in PBS(pH6.2),counted with a haemocytometer and diluted to desired concentrations.Insect bioassays.Bioassays were all done in128-well assay trays(C-D International).Artificial insect diet and T.ni larvae were obtained from the Great Lakes Forestry Centre,Insect Production Service, Ontario,Canada.Assay tray wells contained1ml volumes of diet. OBs were suspended in PBS(pH6.2)and20m l were applied to the diet surface(2.3cm2)and allowed to dry onto the diet surface for1h under shaded rvae were place onto diet,incubated at28u C and mortality data were collected at7days.DNA purification and transfections.Transfections were done with P74D virus DNA and plasmid DNA.Plasmid DNA was purified by CsCl density-gradient centrifugation(Slack&Lawrence,2002).P74D virus DNA was purified from ODVs by methods adapted from O’Reilly et al.(1992).Six150cm2T-flasks,each containing26107 Sf9cells,were infected with AcLP4at a m.o.i.of1p.f.u.per cell and OBs were purified at5days post-infection,cells were harvested and OBs were purified.Suspensions of OBs were pelleted for45min at 1200g and suspended in20ml H2O and were released by the addition of0.5ml of500mM EDTA and2.5ml freshly made2M Na2CO3. After10min,2ml Tris-OH(1M,pH6.8)was added and the insoluble debris was removed at1200g centrifugation for5min.The supernatant was centrifuged at112500g for1h at15u C.The ODV Trypsin cleavage of baculovirus envelope protein P742389pellet was suspended in400m l TE buffer(1mM EDTA,10mM Tris-OH pH8.0)and disrupted by addition of20m l EDTA(500mM), 10m l SDS(10%w/v)and12.5m l proteinase K(10mg ml21).After 2h at37u C,viral DNA was extracted by phenol and chloroform/ isoamyl alcohol(24:1).The DNA was dialysed against TE at4u C in 10K molecular weight cut-off Slide-A-Lyzer cassettes(Pierce). Transfections were done with the transfection reagent,ExGen500 (Fermentas)using methods adapted from Ogay et al.(2006).Sf9cells were seeded onto6-well plates at a density of1.256106cells per well in2ml FBS/Grace’s media.Medium was supplemented with antibiotics/antimycotics(100U penicillin G ml21,100m g streptomy-cin sulfate ml21and250ng amphotericin B ml21)(Sigma-Aldrich). The transfection mixtures included1600ng plasmid DNA,600ng viral DNA,8m l NaCl(1M),1077m l NaCl(150mM)and55m l ExGen500transfection reagent.A transfection mixture volume of 200m l was added to each well and then plates were centrifuged for 5min at500g.At5days post-transfection,cells and media were harvested and processed for either OB purification or SDS-PAGE. Brush border membrane vesicles preparation.BBMV were prepared from80fourth instar T.ni larvae using methods described previously(Slack&Lawrence,2005).Final BBMV preparations were suspended in buffer B(8.5mM MgCl2,150mM D-sorbitol,5mM EGTA,17mM Tris-OH,pH7.4),diluted to1mg ml21total protein and stored at280u C.BBMV total protein was determined with a Bradford reagent assay(Bio-Rad Laboratories).ODV purification and interactions with BBMV.ODVs were produced in Sf9cells that had been co-transfected with P74D virus DNA and P74–EGFP plasmid DNA.Five150cm2T-flasks(1.36108 Sf9cells)were transfected with160m g plasmid DNA and3.6m g P74D virus DNA.DNA was suspended in11ml NaCl(150mM)and550m l ExGen500was added and incubated for15min.The solution was added to110ml FBS/Grace’s media-suspended Sf9cells(1.26106 cells ml21).Cells were then distributed into five150cm2T-flasks (22ml per flask).After5days,OBs were purified as described,but with three additional PBS washes to ensure complete removal of SDS. The final yield was561011OBs per transfection group.ODVs were released from OBs by alkali treatment prior to experiments and were prepared on a small scale in2ml poly-propylene tubes.To a total of1.361010OBs in165m l PBS(pH6.2), 300m l freshly prepared carbonate-buffered potassium was added (CBK)(500mM KCl,100mM Na2CO3,pH10.5).The final solution was incubated at room temperature for5min.Tubes were placed on ice and then centrifuged in an Eppendorf5417R microcentrifuge for 45min,21000g at4u C.The supernatant was aspirated off and ODV pellets remained on ice no more than30min until their suspension in BBMV solutions.Frozen BBMV were thawed on ice and then diluted50%in CBK buffer.The resulting BBMV incubation(BI)buffer contained 250mM KCl,50mM Na2CO3,75mM D-sorbitol,1.25mM EGTA, 4mM Tris-OH and4mM MgCl2.The pH of BI buffer was10.5.In some experimental groups,BBMV were either preheated to95u C for 5min or type II SBTI(Sigma-Aldrich)was added to a final concentration of500m g ml21.BBMV were further diluted in BI buffer to desired concentrations of BBMV proteins and55m l volumes of the vesicles were added to the ODV pellets.Control groups just received BI buffer.ODV pellets were suspended by brief vortexing and incubated for20min at28u C and then placed on ice.This was followed by the addition of55m l26Laemmli SDS-PAGE disruption buffer and heating at95u C for10min.Western blots.Proteins were fractionated under reducing condi-tions in8.75%w/v acrylamide:bis(37:1)SDS-PAGE gels and transferred to nitrocellulose.Western blotting was done using an enhanced chemifluorescence(ECF)Western blotting kit(AmershamBiosciences).Primary antibodies were either the GP64-specific mono-clonal antibody,AcV5(Hohmann&Faulkner,1983)or a EGFP-specificmonoclonal antibody JL-8(Clontech).Blots were blocked overnight at 4u C with10%w/v powdered milk in PBS(pH7.4)and were probedfor2h at room temperature with either AcV5or JL-8diluted1:500inPBS(pH7.4),0.05%v/v tween-20(PBS-T).Blots were probed for2hwith secondary anti-mouse,fluorescein conjugated antibody at1:500 dilution in PBS-T and then probed for2h with tertiary anti-fluorescein,alkaline phosphatase(AP)conjugated antibody at1:2000dilution in PBS-T.After tertiary antibody probing,the blots were washed briefly with1mM MgCl2,10mM Tris-OH(pH9.5)and thenreacted with1.25m g ml21dimethylacridinone phosphate(MolecularProbes)fluorescent substrate.Blots were scanned using a TyphoonTrio+laser bed scanner(GE Healthcare Life Sciences)(633nm excitation,670nm BP30nm emission).Protein sequence analysis and sequence sources.Alignment ofthe amino acid sequences of the P74homologues was done using MEGALIGN5.08(DNASTAR).The CLUSTAL W method(Thompson et al., 1994)and Gonnet250protein weight matrix were used in alignments(gap penalty10,gap length penalty0.2).Hydrophobicity profile(15aa window)was determined using theprogram Protean5.08(DNASTAR).For transmembrane(TM)domainprediction,the site http://www.cbs.dtu.dk/services from the Centerfor Biological Sequence Analysis at the Technical University of Denmark was accessed and the program TMHMM2.0(Krogh et al., 2001)was used.RESULTSRole of trypsin in the infectivity of lepidopteran baculovirusesP74is essential for per os infection and is cleaved at a specific location by lepidopteran midgut trypsins.As a model system for other baculoviruses,we wanted to ascertain the role of indigenous midgut trypsin in per os infectivity.The experiments involved feeding T.ni larvae with parental AcMNPV OBs along with either SBTI or trypsin.Varying amounts of OBs were fed to groups of insects such that lethal concentration for50%(LC50) values could be estimated.It was observed that,relative to control groups of insects that were fed only OBs,the SBTI increased the LC50threefold and the presence of trypsin decreased the LC50fivefold(Fig.1).Differences in the LC50 in the presence of either additives clearly demonstrate that trypsin plays an important role in virus infectivity.Development of a transient expression-based assay for characterization of P74A simple method to evaluate P74mutants without constructing new recombinant viruses was developed.It involved transfecting cells with plasmids that transiently express P74in the presence of a co-infecting P74D virus. This was similar to a successful approach involving p74-expressing insect cell lines complementing a P74D virus (Wilson,1997).We used pBAC-5-based plasmids to transiently express the p74ORF fused in-frame with the 59end of the EGFP ORF(Fig.2a).These plasmids wereJ.M.Slack and others2390Journal of General Virology89previously used to study P74localization in infected cells (Slack et al.,2001).Though designed as baculovirus shuttle vectors,pBAC-5-based plasmids are excellent for transient gene expression in insect cell culture (Ogay et al.,2006),due to the presence of an AcMNPV gp64early/latepromoter (Whitford et al.,1989;Blissard &Rohrmann,1991).Homologous recombination of plasmids such as pBAC-5with circularized viral DNA is an infrequent event (less than 1%)(Kitts et al.,1990)and thus would not be significant for these transient experiments.Also,pBAC-5plasmids are designed to eliminate the polh gene after recombination and any incidental recombinant viruses would not produce the polyhedra that were used to perform experiments.The C-terminal tagging of P74with EGFP enabled monitoring of transfections and also provided an epitope tag for immunodetection (Fig.2b).The OBs produced from P74D virus/P74–EGFP plasmid co-transfections appeared by microscopic examination to be similar to parental virus OBs.Western blotting confirmed that P74–EGFP proteins co-purified with ODVs (Fig.2b,ODV lane).More importantly,the P74D virus was rescued for per os infectivity by P74–EGFP,but not by EGFP (Table 1).Trypsin cleavage site mutantsThe previous hypothesis of midgut-specific P74cleavage at R156(Slack &Lawrence,2005)was based on the estimated size of tryptic digest fragments in Western blots and on conservation of the R156trypsin cleavage site among P74homologues.It was not possible to obtain enough material to do N-terminal sequencing of P74trypticdigestFig. 1.Effects of trypsin and SBTI on susceptibility of T.ni larvae to AcMNPV infection.Second instar T.ni larvae were fed dosages of AcMNPV OBs in the presence of trypsin or SBTI and compared to OBs alone (control).Test animals received 1mg/diet well porcine parvovirus tested (1:250)trypsin or 1mg/diet well type II SBTI.(a)Cumulative %mortality is plotted against OB amounts that were added to diet wells.(b)The LC 50of each group is graphed and was determined by probit analysis using the program PoloPlus1.0(LeOra Software).Numerical bioassay data are provided in Supplementary TableS2.Fig. 2.Control co-transfection experiments.Initial experiments involved co-transfection of the P74D virus DNA with plasmid DNA expressing either EGFP or p74–EGFP .(a)The EGFP and P74–EGFP ORFs are schematically illustrated along with the predicted molecular masses of their products.(b)We confirmed the synthesis of EGFP and P74–EGFP by Western blotting with an anti-EGFP ne M is mock-infected Sf9cells,lane 1is cells transfected with P74D virus DNA only,lane 2is cells transfected with pBAC-5-EGFP and P74D virus DNA,lane 3is cells transfected with pBAC-5-p74-EGFP and P74D virus DNA.The lane labelled ‘ODV’is purified ODVs from cells that had been co-transfected with pBAC-5-p74-EGFP and P74D virus DNA.The positions and molecular masses in kDa of markers are indicated on the sides.Table 1.Mortality of 2nd instar T.ni larvae 7days post feeding OBs (100000OBs per well)from P74D virus,P74D virus/plasmid co-transfections or wt AcMNPVVirus Plasmid Mortality (dead/total)P74D AcMNPV 20/16P74D AcMNPV EGFP 0/16P74D AcMNPV p74–EGFP16/16AcMNPV 216/16Mock20/16Trypsin cleavage of baculovirus envelope protein P742391fragments and site-directed mutagenesis was chosen to verify if R156or nearby trypsin cleavage sites are required for P74function.In the vicinity of R156,there are11 consensus trypsin cleavage site motifs(R^X,K^X except R^P,K^P)(Fig.3)and all are present in AcMNPV P74at R114,R119,R132,K138,K147,R156,R184,K186,R195, R196and R199.A series of sited-directed mutants were constructed by substituting R or K residues with glutamine(Q)residues. Glutamine was chosen because its side chain size is similar to R or K residues.Glutamine is a polar amino acid residue and R and K are positively charged residues.However,in the highly alkaline conditions of the insect gut where P74is to function,K and R residues would also become polar residues.Also,this Q substitution occurs naturally in some P74homologues at K138and R186(Fig.3).In the first group of mutants,each of the residues R114,R119,R132, K138,K147and R156were changed to Q residues.It was initially opted to make multiple mutations where trypsin cleavage sites were clustered close to each other(R184/ K186and R195/R196/R199).Site-directed mutants were made in P74–EGFP plasmids that were then co-transfected with P74D virus DNA into insect cells.It was confirmed by Western blot with EGFP-specific monoclonal antibody that similar amounts of P74–EGFP were being translated from each mutant P74–EGFP plasmid(Fig.4a).As an internal control to virus replication,Westerns were also probed with anti-GP64 monoclonal antibody,AcV5(Hohmann&Faulkner,1983; Monsma&Blissard,1995).OBs from each co-transfection group were fed to2nd instar T.ni larvae at several dosages.The bioassay results of the first group of P74mutants are plotted as cumulative mortality(Fig.4c).It was evident from this experiment and others that most of the P74–EGFP mutants rescued per os infection of the P74D virus as effectively as the native P74–EGFP protein.R156was not required for P74’s function in oral infection.One clear and interesting observation is that cells producing the multiple site P74mutant R195Q/ R196Q/R199Q resulted in OBs that were1000-fold less infectious than OBs derived from the other groups and warranted furtherinvestigation.Fig.3.Alignment of predicted trypsin cleavage sites.A portion of the N terminus of AcMNPV P74was aligned with other P74 homologues.The consensus sequence(Cons)is shown at the top.The R and K residues of predicted trypsin cleavage sites are highlighted.P74homologues are arranged into group I alphabaculoviruses(I),group II alphabaculoviruses(II), betabaculoviruses(B),hymenopteran-specific gammabaculoviruses(G),the dipteran-specific deltabaculovirus(D),Cuni and Hz-1.The group is indicated on the right side.The numbers along the top correspond to amino acid positions of AcMNPV P74.For this alignment,short form virus source names are indicated on the left side.Ac/Px,Cf/Op,Hear/Hz,and Co/Cf GV are baculovirus species with P74homologues that are identical in the region shown and are represented by single sequences.Also Splt(2)and Sf(2)indicates there are two virus isolates with P74homologues that are identical in the region shown.P74 homologue source baculovirus full names and GenBank protein accession numbers are listed in Supplementary Table S5.J.M.Slack and others2392Journal of General Virology89P74site-directed mutants in region R195/R196/R199The multiple mutations in this region of the protein could have caused conformational changes that precluded the ability of the mutant to rescue infectivity.Also,the P74D virus used in these experiments contained a p74gene region corresponding to M1to C194.It is possible homologous recombination of our p74–EGFP plasmids with P74D virus DNA rescued P74mutations between R114and K186and that mutations beyond C194were not.We,therefore,generated a second series of site-directedmutants that covered all combinations of mutations in the R195/R196/R199region.Bioassays were carried out as previously described and the results are summarized in Fig.4(d).The single mutants R195Q,R196Q and R199Q were as functional as native P74–EGFP and could rescue per os infectivity of the P74D virus.The double mutants R195Q/R196Q and R195Q/R199Q were also able to rescue per os infectivity.However,the double mutant R196Q/R199Q produced OBs that were at least 100-fold less infectious than the native P74–EGFP group.These data suggestthatFig.4.Bioassay of P74site-directed mutants.Sf9cells were co-transfected with P74D virus DNA and the p74–EGFP plasmid or p74–EGFP plasmids with mutations in the P74ORF.Numbers refer to the amino acid position where R or K residues were changed to Q residues.The control group was just P74D virus DNA.(a and b)Western blotting was done with two different antibodies to confirm the presence of P74–EGFP proteins or to detect the virus-specific protein,GP64.The blots were probed with either anti-GFP monoclonal antibody (a -GFP)or with the AcV5anti-GP64monoclonal antibody (a -AcV5).Antibody reacting portions of blots are shown in upper two panels (102.6kDa P74–EGFP and 64kDa GP64).Also in the lower panel of (a)and (b),SDS-PAGE gels were stained with Coomassie brilliant blue and the dominant staining 29kDa protein which is likely to be polyhedrin is shown (Polh).(c and d)In two separate bioassays,purified OBs from mutant groups were fed to 2nd instar T.ni larvae at varying dosages.The cumulative mortality curve is plotted.Most P74–EGFP mutants rescued per os infectivity of the P74D virus like wt P74–EGFP except for mutants P74(R195Q/R196Q/R199Q)-EGFP (c and d)and P74(R196Q/R199Q)–EGFP (d).Numerical bioassay data are provided in Supplementary Tables S3and S4.Trypsin cleavage of baculovirus envelope protein P742393R196and R199may be functionally redundant for the per os infectivity.None of the mutants compromised P74function as much as mutant R195Q/R196Q/R199Q,indicating that the three R residues may work in concert.BBMV-specific cleavage of P74BBMV from T.ni larvae were used to evaluate the effects of insect trypsins on P74.By using azocasein assays (Slack &Lawrence,2005)and different proteinase inhibitors,it was determined that BBMV from T.ni contained almost entirely trypsin protease activity (data not shown).In experiments,P74–EGFP or mutant P74(R195Q/R196Q/R199Q)–EGFP-containing ODVs were purified and incu-bated with BBMV,followed by fractionating the proteins on SDS-PAGE.P74–EGFP cleavage products were detected by Western blot using an anti-EGFP antibody (Fig.5).BBMV-specific trypsin cleavage of native P74–EGFP was compared with that of the mutant P74(R195Q/R196Q/R199Q)–EGFP.After incubation with BBMV,the 102.6kDa native P74–EGFP protein produced a cleavage product of 80kDa (Fig.5,lanes 9and 11)whereas the mutant P74–EGFP protein did not produce this product (Fig.5,lanes 10and 12).The presence of SBTI inhibitedthe cleavage of P74–EGFP by the BBMV (Fig.5,lanes 7and 8).Although our ODV preparations were partially purified,these experiments suggest that P74–EGFP cleavage occurs in the context of the ODV and that the cleavage takes place at residues R195,R196and R199.DISCUSSIONThe data presented here clearly suggest that trypsin activates the per os infectivity of baculovirus ODVs by cleaving P74at the R195/R196/R199vicinity.Tryptic activation has been documented for other viruses including coronaviruses (Frana et al.,1985),rotaviruses (Vonderfecht et al.,1988),Sendai virus (Muramatsu &Homma,1980)and poxviruses (Ichihashi &Oie,1982).The lepidopteran midgut is rich in trypsins (Johnston et al.,1991;Terra &Ferreira,1994;Oliveira et al.,2005;Pereira et al.,2005)and it is probable that baculoviruses have evolved to exploit this environment.Trypsin is a serine protease and,in past studies,serine proteases have been found associated with insect-derived baculovirus OBs (Eppstein &Thoma,1975;Eppstein et al.,1975;Langridge &Balter,1981;Maeda et al.,1983).Site-directed mutagenesis data and BBMV cleavage data suggest that P74is a target of midgut trypsins and point to R196and R199being alternate primary trypsin cleavage sites on P74.So long as R196or R199are present,the full P74function is retained.Residues R196and R199are present among P74homologues of alphabaculoviruses,betabaculoviruses and gammabaculoviruses.The only exception is one isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus which has a P74homologue lacking R196(Sf-CV,Fig.3).There was insufficient material for N-terminal sequencing of BBMV-specific P74–EGFP cleavage products,but the molecular masses of the products correspond well with cleavage in the R195/R196/R199region.This is supported by lack of a specific cleavage product in the R195Q/R196Q/R199Q mutant.Two studies (Haas-Stapleton et al.,2004;Yao et al.,2004)have concluded that P74is a viral attachment protein.Haas-Stapleton et al.(2004)showed that the P74D virus could not compete with wt virus ODVs for binding to the midgut and Yao et al.(2004)identified a 30kDa midgut receptor protein for P74.P74cleavage by midgut proteases may expose a midgut receptor binding domain on P74.However P74binds to BBMV in the presence of serine protease inhibitors (Yao et al.,2004)and thus trypsin cleavage of P74is not prerequisite to virus attachment.P74has characteristics of proteolytically activated virus envelope proteins such as influenza virus haemagglutinin (HA)protein and paramyxovirus F protein.Like P74,these proteins are C-terminally anchored in the virion envelope and have a large surface-exposed N-terminal domains.When influenza virus HA proteins and paramyxovirus F proteins are cleaved by host trypsins,a hydrophobic membrane insertion N-terminal domain isexposedFig. 5.Western blot of ODV-associated native P74or P74mutant R195Q/R196Q/R199Q after incubation with BBMV.An experiment was done to examine midgut BBMV-specific proteo-lytic cleavage of wt P74–EGFP and P74mutant (R195Q/R196Q/R199Q)–EGFP in context of the ODV.OBs were purified from Sf9cells that had been co-transfected with P74D virus DNA and either P74–EGFP (wt)or P74(R195Q/R196Q/R199Q)–EGFP (MT)plasmids.The ODVs were released from OBs and incubated with varying amounts of BBMV prepared from T.ni larvae midguts.Proteins were then fractionated by SDS-PAGE and Western blotted with a -EGFP monoclonal antibody.The result is shown here.The positions of molecular mass standards are indicated on the ne 1is the BBMV alone and lane 2is the P74D virus ODVs alone.The 3rd and 4th lanes are wt and MT P74–EGFP-containing ODVs incubated alone.We incubated ODVs in BBMV that had been previously boiled (5th and 6th lanes)or treated in SBTI (7th and 8th lanes).Lanes 9through 12are ODVs incubated with two different amounts of BBMV.In lanes we indicate the amount of BBMV used in incubations as the number of ng of BBMV-specific protein.The positions of full-length P74–EGFP (102.6kDa)and a cleavage product of interest (80.0kDa)are indicated on the right side.J.M.Slack and others2394Journal of General Virology 89。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
杆状病毒关键词：昆虫病毒，杆状病毒，核型多角体病毒，颗粒体病毒，质型多角体病毒杆状病毒是一类在自然界中专一性感染节肢动物的DNA病毒，病毒粒子呈杆状，基因组为双链环状DNA分子，DNA以超螺旋形式压缩包装在杆状衣壳内，大小在90～180 Kb之间。
目前杆状病毒作为高效、安全的无公害生物虫剂广泛应用于害虫防治。
杆状病毒只来源于无脊椎动物，虽然已发现600多种杆状病毒，但进行分子生物学研究的不到20种。
杆状病毒的基因组为单一闭合环状双链DNA 分子，大小为80～160 kb，其基因组可在昆虫细胞核复制和转录。
DNA复制后组装在杆状病毒的核衣内，后者具有较大的柔韧性，可容纳较大片段的外源DNA 插入，因此是表达大片段DNA的理想载体。
其中，用作外源基因表达载体的杆状病毒，目前仅限于核型多角体病毒(nuclear polyhedrosis virus，NPV)。
该病毒颗粒在细胞内可由多角体蛋白包裹形成长度约1~5 m的包含体病毒，呈多角体形状。
核型多角体病毒有两种形式：一种为包含体病毒(occluded virus，OV)，另一种则为细胞外芽生病毒(budded virus，BV)。
它们在病毒感染中扮演的角色不同，包含体病毒是昆虫间水平感染的病毒形式，昆虫往往是食入污染OV的食物后引起感染。
包含体病毒外层裹了一层蛋白晶体，即为29 000的多角体蛋白，它对病毒的水平感染起以下作用：①保护病毒颗粒在外界传播过程中免遭环境因素的破坏而失活。
②保证病毒颗粒在适当的位置释放，引起感染。
昆虫中肠上皮局部的强碱性环境(pH=10.5)，可使病毒颗粒释放蛋白酶溶解多角体。
BV病毒是个体内细胞间的感染形式，由细胞芽生出BV，进入血淋巴系统中感染其它部位的细胞或直接在临近细胞内感染。
近几十年，有关杆状病毒基因结构、功能和表达调节的研究进展迅速，其中研究最深入的是mùxu苜蓿银蚊夜蛾(autogra—phacalifornica)多核型多角体病毒(multiple nuclear polyhedro-sis virus，MNPV)，简称AcMNPV或AcNPV。
该病毒是杆状病毒科 Baculoviridae的原型，是一种大的、带外壳的双链DNA病毒，能感染30多种鳞翅目昆虫，被广泛用作基因表达系统载体。
其它作为表达载体的杆状病毒，主要是来自家蚕的NP~(bombyx moil，BmNP~)。
由于家蚕幼虫体内系统适合大规模地制备生产外源蛋白，且成本低，显示出良好的应用前景。
本文主要介绍 AcNPV病毒，BmNPV在许多方面与其具有共同的特征。
AcNPV的基因表达分为4个阶段：立即早期基因表达、早期基因表达、晚期基因表达和极晚期基因表达。
前两个阶段的基因表达早于DNA复制，而后两个阶段的基因表达则伴随着一系列的病毒DNA合成。
其中在极晚期基因表达过程中，有两种高效表达的蛋白，它们是多角体蛋白和P10蛋白：多角体蛋白是形成包含体的主要成分，感染后期在细胞中的积累可高达30％~50％，是病毒复制非必需成分，但对病毒粒子却有保护作用，可使之保持稳定和感染能力另一类高效表达的极晚期蛋白为P10蛋白，也是一类病毒复制非必需成分，可在细胞中形成纤维状物质，可能与细胞溶解有关。
多角体基因和P10基因现在都已被定位和克隆这两个基因的启动子具有较强的启动能力，因此这两个基因位点成为杆状病毒表达载体系统理想的外源基因插入位点。
1.杆状病毒基因组的结构和功能研究杆状病毒基因组为双链环状DNA分子。
DNA以超螺旋方式被压缩包装在杆状核衣壳(rod.shaped nueleocapsid)内，核衣壳包被脂质蛋白囊膜(envelope)后形成病毒粒子。
核衣壳包括衣壳(capsid)蛋白和髓核(COle)。
其中衣壳蛋白是杆状病毒粒子的主要结构蛋白；髓核由病毒DNA分子和与其密切相关的碱性蛋白构成。
碱性蛋白同DNA紧密结合以维持其复杂有序的超螺旋结构。
目前已知基因组全序列的杆状病毒有苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)b]、家蚕核多角体病毒(BmNPV) 、黄杉毒蛾多核衣壳核多角体病毒(OpMNPV)、舞毒蛾多核衣壳核多角体病毒(LdMNPV) 、甜菜夜蛾多核衣壳核多角体病毒(SeMNPV)、棉铃虫核型多角体病毒(HaNPV)，以及斜纹夜蛾核型多角体病毒(SphMNPV)。
目前AcMNPV基因组的研究最为深入，它是双链超螺旋大分子环状DNA，其大小为90～160kb，约编码154个基因。
AcMNPV基因组的基因组织(gene organization)较为复杂，基因组的不同区域具有功能分化，基因的分布尚无规律可循，但AcMNPV基因组含有8个同源区，每区含有不等的重复或倒置重复序列，这些重复序列由位于中间的不完整回文序列及其两侧各约20bp的序列构成。
同源区是杆状病毒基因组普通存在的功能域结构，对于基因表达的调节具有增强作用，同时也是DNA复制的原点。
杆状病毒中现已鉴定的基因近70种，可分为结构蛋白基因和非结构蛋白基因两大类。
结构蛋白基因如polh、P10、gp64、p6.9、gp41、vp39等基因。
非结构基因中，与DNA复制相关的重要基因有helicase基因(he1)、dnapol、lef-1、lef-2、lef-3、ie-1、/ie-2、p35、pe-38等；起表达调节作用的基因主要有ie-1、ie-2、lef类基因、p35、pe38等。
这些代表性基因与其功能的关系见表1。
2．杆状病毒载体表达系统的特点AcNPV病毒用作外源基因的表达载体，通常是通过体内同源重组的方法，用外源基因替代多角体蛋白基因而构建重组病毒。
由于多角体基因启动子在感染后18~24h开始转录和翻译，一直持续到70 h。
外源基因置换掉多角体基因后，并不影响后代病毒的感染与复制，意味着重组病毒不需要辅助病毒的功能。
杆状病毒表达系统自从第一次用来表达干扰素以后在许多重组蛋白的表达中得到广泛应用，例如用于表达白介素(IL)一2，3、BMP及多种病毒蛋白等。
相对其他表达系统它具有以下几个方面的特点：①组蛋白具有完整的生物学功能：杆状病毒表达系统可为高表达的外源蛋白在细胞内进行正确折叠、二硫键的搭配及寡聚物的形成提供良好的环境，可使表达产物在结构及功能上接近天然蛋白。
②②能进行翻译后的加工修饰：杆状病毒表达系统具有对蛋白质完整的翻译后加工能力，包括糖基化、磷酸化、酰基化、信号肽切除及肽段的切割和分解等，修饰的位点与天然蛋白在细胞内的情况完全一致：对比实验证明，在昆虫细胞发生的糖基化位点与哺乳动物细胞中完全一致，但修饰的寡糖种类却不完全一样。
这种不一致对不同目的蛋白的活性影响不同，所以昆虫表达系统还可作为一个研究糖基化对蛋白质结构与功能影响方面的理想模型。
③③表达水平高：与其它真核表达系统相比较，此系统最突出的特点就是能获得重组蛋白高水平的表达，最高可使目的蛋白的量达到细胞总蛋白的50％。
④能容纳大分子的插入片段：杆状病毒毒粒可以扩大，并能包装大的基因片段，但目前尚不知杆状病毒所能容纳的外源基因长度的上限。
④⑤能同时表达多个基因：杆状病毒表达系统具有在同一细胞内同时表达多个基因的能力。
既可采用不同的重组病毒同时感染细胞的形式，也可在同一转移载体上同时克隆两个外源基因，表达产物可加工形成具有活性的异源二聚体或多聚体。
另外，昆虫杆状病毒表达系统具有剪切的功能，能表达基因组DNA；还有对重组蛋白进行定位的功能，如将核蛋白转送到细胞核上，膜蛋白则定位在膜上，分泌蛋白则可分泌到细胞外等。
最后，杆状病毒对脊椎动物无感染性，现有研究也表明其启动子在N-％动物细胞中没有活性，因此在表达癌基因或有潜在毒性的蛋白时可能优于其它系统。
⑤3．杆状病毒载体的重组与筛选杆状病毒由于基因组庞大，外源基因的克隆不能通过酶切连接的方式直接插入，必须通过转移载体的介导．即将极晚期基因(如多角体基因及其边界区)克隆入细菌的质粒中，消除其编码区和不合适的酶切位点，保留其5’端对高效表达必需的调控区，并在其下游引入合适的酶切位点供外源基因的插入，即得到转移载体。
将要表达的外源基因插入其启动子下游，再与野生型AcNPV DNA共转染昆虫细胞，通过两侧同源边界区在体内发生同源重组，使多角体蛋白基因被外源基因取代。
而将外源基因整合到病毒基因组的相应位置，由于多角体基因被破坏，则不能形成多角体。
这种表型在进行常规空斑测定时，可同野生型具有多角体的病毒空斑区别开来，这就是最初的筛选重组病毒的方式。
但由于重组效率较低(0.1％-1％)，表型差别不显著，应用上有一定的困难。
为此，经过不断探索，在重组杆状病毒的筛选与鉴定方面取得了很大改进，具体方法有以下几种。
3.1．半乳糖苷酶的蓝白筛选1990年，Vialard等在多角体基因的上游，利用pl0基因启动子带动LacZ基因构建了转移载体pJVNheI。
将其共转染sf细胞后，重组病毒可表达B-半乳糖苷酶，通过加入x-gal使之形成蓝色空斑，便可进行重组病毒的筛选。
1990年，Kins提出了线形化技术，其原理是线形化的杆状病毒基因组感染性很低，但仍具有与引入细胞内的同源序列进行同源重组的能力。
如果同源序列位于线形化杆状病毒的两端，则基因组即可环化恢复完整的感染性，使阳性重组率大大提高。
蓝白斑筛选蓝白斑筛选是一种基因工程常用的重组菌筛选方法。
野生型大肠杆菌产生的β-半乳糖苷酶可以将无色化合物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)切割成半乳糖和深蓝色的物质5-溴-4-靛蓝。
有色物质可以使整个培养菌落产生颜色变化，而颜色变化是鉴定和筛选的最直观有效的方法。
设计适用于蓝白斑筛选的基因工程菌为β-半乳糖苷酶缺陷型菌株。
这种宿主菌的染色体基因组中编码β-半乳糖苷酶的基因突变，造成其编码的β-半乳糖苷酶失去正常N段一个146个氨基酸的短肽（即α肽链），从而不具有生物活性，即无法作用于X-gal产生蓝色物质。
用于蓝白斑筛选的载体具有一段称为lacz'的基因，lacz'中包括：一段β-半乳糖苷酶的启动子；编码α肽链的区段；一个多克隆位点(MCS)。
MCS位于编码α肽链的区段中，是外源DNA的选择性插入位点，但其本身不影响载体编码α肽链的功能活性。
虽然上述缺陷株基因组无法单独编码有活性的β-半乳糖苷酶，但当菌体中含有带lacz'的质粒后，质粒lacz'基因编码的α肽链和菌株基因组表达的Ｎ端缺陷的β-半乳糖苷酶突变体互补，具有与完整β-半乳糖苷酶相同的作用X-gal生成蓝色物质的能力，这种现象即α-互补。
操作中，添加IPTG(异丙基硫代-β-Ｄ-半乳糖苷)以激活lacz'中的β-半乳糖苷酶的启动子，在含有X-gal的固体平板培养基中菌落呈现蓝色。