杆状病毒-ppt课件
对虾杆状病毒病
组织损伤
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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。
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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。
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对虾杆状病毒病
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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
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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。
杆状病毒资料
DOCS
DOCS SMART CREATE
杆状病毒：基本概念与深入研究
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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
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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。
杆状病毒-课件
杆状病毒杀虫剂的研究
杆状病毒杀虫剂作为农作物、森林害虫防治 的新型安全的生物农药，已经得到了人们的重视。 与传统的化学农药相比，杆状病毒对宿主昆虫具 有高度的病原性，对非靶生物十分安全，能够在 环境中长期存活，对人、畜及其他脊椎动物无害， 不污染环境，能和其他化学农药混合使用，同时 具有防效时间长、使用方便等优点，所以早在 1973年就已被联合国粮农组织和世界卫生组织推 荐作为化学农药的理想替代品。
除了和复制相关的基因以外, 在AcMNPV基因组
中共鉴定出10个基因与晚期基因的转录有关,它们是lef4, lef-5, lef-6, lef-8, lef-9, lef-10, lef-11, lef-12, 39K和p47等 基因。其中4个保守基因( lef-4, lef-8, lef-9和p47)的产物
病毒基因表达
早期基因的表达 早期时相向晚期基因表达的过渡 晚期基因的表达
早期基因的表达
早期基因的转录是由宿主RNA聚合酶Ⅱ 介导的。早期基因迅速表达以满足病毒复制 周期所需条件：
控制宿主细胞的代谢机器 为病毒DNA复制提供必需条件 突破或阻断宿主细胞防御机制 提供下一时相病毒基因表达所需基因产物
HaSNPV ）
杆状病毒的分类
Baculoviridae 杆状病毒科
NPV Nucleopholyhedrovirus 核型多角体病毒属
GV Granulovirus 颗粒体病毒属
MNPV SNPV
Baculoviridae
NPV GV
组I 组 II
AcMNPV OpMNPV BmMNPV
LdMNPV SeMNPV HaSNPV
杆状病毒载体-演示文稿
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。
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。
⑤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。
❖ 杆状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。
重组杆状病毒表达载体的结构
达产物，将其致敏原性降到最低限度是值得重视和探讨的问题。 (4)由于该系统独特的性质，使其被广泛地应用于基因工程、药 物开发、疫苗生产、表达免疫活性分子和某些致瘤病毒蛋白以及 基因表达调控研究等多个领域中。迄今为止，已有数百个基因在 昆虫细胞或幼虫体内得到高效表达，为获得大量的类原型蛋白及 其功能研究提供了可能。
家蚕核型多角体病毒粒子模式图
❖ 杆状病毒粒子呈杆状， 大小为330×80nm。由 膜及核髓组成。膜为三 片层状，外层பைடு நூலகம்由蛋白 质白质及脂质组成的套 膜，现代病毒学称为囊 膜，内层是由蛋白质构 成的衣壳，中间为胶粘 层。衣壳内为髓核，是 呈螺旋型的双链脱氧核 糖核酸分子。衣壳和髓 核构成核衣壳。在一个 套膜中，包裹的核衣壳 数目因寄生的部位不同 而异。
昆虫杆状病毒杀虫剂课件
苏云金杆菌复配等。
•昆虫杆状病毒杀虫剂
•17
结语
昆虫杆状病毒杀虫剂具有对人、畜和环境安全，
不易产生抗性等优点，且符合IPM农业可持续发展
的要求。以开发得最为成功的棉铃虫病毒杀虫剂为
例，至今的十几年间，累计生产逾2000t，推广面
积超过66.67万公顷。但是，我国各类生物农药的
个方面：
⑴修饰病毒本身基因以提高杆状病毒的感染力;
⑵在杆状病毒基因组中插入外源基因以增强病
毒毒力（研究较多）;
⑶用异源病毒重组来扩大杆状病毒杀虫谱。
•昆虫杆状病毒杀虫剂
•13
⑴修饰病毒本身基因
•
e.g.缺失egt基因
egt基因是杆状病毒在个体水平调控感染宿主生长发育
的唯一已知基因。缺失egt基因的重组病毒可引起幼虫生长
发育代谢的失调, 加速感染虫体的死亡。
OReily等(1991)发现银纹夜蛾核多角体病毒的一个早期
非必需基因蜕皮甾体尿苷二磷酸葡萄糖转移酶基因, 该基因
受早期启动子控制, 编码的一个506aa蛋白能阻止幼虫蜕皮
和化蛹, 促进病毒自身的增殖。缺失该基因的重组病毒能比
野生型病毒提早27.5h杀死粉纹夜蛾幼虫, 受感染的幼虫取食
量也减少了40% 。
•昆虫杆状病毒杀虫剂
•14
⑵插入外源基因
e.g.插入植物蛋白酶抑制剂基因
植物蛋白酶抑制剂基因编码的植物蛋白酶抑制
因子作为植物抵抗害虫的天然防御体系, 近年来已
引起人们的注意。季平等(1996)把慈姑蛋白酶抑制
剂B基因插入家蚕BmNPV 基因组中, 获得了带慈姑
蛋白酶抑制剂B基因的重组家蚕BmNPV。重组病毒
杆状病毒表达系统介导的猪圆环病毒2型基因工程疫苗的研究.PPT课件
摘要
• 猪2型圆环病毒(Porcine circovirus type 2, PCV2) 可单独感染，也可继发感染，近几年在世界各地 普遍流行,是对养猪业危害很大的一类疾病。它可 引起仔猪断奶后多系统衰竭综合征(PMWS)、猪 皮炎和肾病综合病(PDNS)、繁殖障碍、猪呼吸综 合征(PRDC)、仔猪先天性震颤、增生性/坏死性 肠炎(PNP)、“无名高热症”等。在临床上PCV2普 遍存在免疫抑制和免疫激活现象，降低猪的免疫 力，使猪生长缓慢，造成其他疾病的大爆发。
• 2、杆状病毒介导的ORF2亚单位疫苗的构建及免疫原性研 究： • 人工合成PCV2 ORF2基因，构建转移载体pFastBacDual2ORF2。将此载体转化DH10Bac大肠杆菌感受态细胞， 经蓝白斑筛选后获得穿梭质粒Bacmid-2ORF2。将此质粒 转染Sf9细胞，获得杆状病毒Ac-2ORF2。将此杆状病毒转 染Sf9昆虫细胞后，ORF2基因可在细胞中大量表达。将表 达的ORF2蛋白制备成亚单位疫苗，分三个剂量：106个 cell/mL、5×106个cell/mL和107个cell/mL免疫4～6周龄的 BALB/c小鼠。试验结果表明：首免三周后，三个免疫组 106个cell/mL、5×106个cell/mL和107个cell/mL小鼠体内 已有ORF2的ELISA抗体和中和抗体产生，加强免疫后此 三组的抗体水平继续升高，其中5×106个cell/mL最高， ELISA抗体滴度可达到1：2133，中和抗体滴度可达到1： 8.33。表明此杆状病毒表达的ORF2蛋白有较好的免疫原 性，可以作为PCV2的一种新型的候选业单位疫苗。
BglⅡ识别位点是：A'GATCT BamHⅠ识别位点是：G'GATCC
昆虫表达系统 ppt课件
转录病毒感染法
PPT课件
13
表达产物的检测技术
Western印迹法——免疫学方 法——抗原-抗体反应
荧光显微镜法——表位标记的 抗体与直接抗体进行荧光标 记——免疫学方法
不影响蛋白功能
基因，以利于产生天
有利于研究蛋白功能
然蛋白
常用表位：6His；FLAG； LZ等
其他常规构建载体的 注意点
PPT课件
12
基因导入的各种方法
光穿孔法：激光；悬浮细胞 冲击波：活体局部的基因转移 基因枪法：即微粒轰击；动物细胞，更适用于植
物细胞 穿孔法：脉冲电场；Cytomix缓冲液；70%细胞
PPT课件
3
病毒复制（SV40示例）
• 以单纯疱疹病毒为例。 （1）病毒与细胞结合； （2）病毒进入细胞，去 包膜；（3）脱壳；（4） 病毒DNA进入细胞核； （5）病毒基因组复制， 合成子代病毒及病毒 mRNA；（6）以病毒基因 转录的mRNA进入细胞质； （7）病毒mRNA翻译病毒 子代蛋白，包括早期蛋白 和晚期蛋白；（8）装配 子代病毒；（9）出核， 同时披上包膜；（10）释 放到胞外
• 在杆状病毒所表达的一系列蛋白中,有一类蛋白具有较高的表达量,并且均为病 毒基因组复制所非必需, 其中最具代表性的有多角体蛋白与 P10蛋白, 均属于 晚期表达的蛋白,受晚期启动子的调控。昆虫的杆状病毒表达系统正是利用了 这类蛋白的优点, 从而提供了外源 DNA插入座位。
PPT课件
10
杆状病毒表达系统优点
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艾滋病的概述
参考文献 曾毅：爱滋病的流行趋势、研究进展及遏制策略 联合国2008全球艾滋病流行状况报告，中国感染人数上 升 医学、营养药膳学：营养干预可明显延缓艾滋病发病 艾滋病的感染治疗和预防 解析艾滋病"获得性免疫缺陷综合症"： 艾滋病检测中心文献 艾滋病检测中心 中医治疗艾滋病 洁爱网
微生物教研组 郑方亮
杆状病毒简介
杆状病毒是节肢动物的病源病毒，其天 然宿主主要是鳞翅目、膜翅目和双翅目的 昆虫， 还有少数杆状病毒可以感染甲壳纲 的节肢动物，目前已从超过800种昆虫体内 分类鉴定出了600多种杆状病毒。
棉铃虫
棉铃虫单粒包埋核型多角体病毒 （Helicoverpa armigera singlenucleocapsid nucleopolyhedrovirus， HaSNPV ）
杆状病毒其它功能基因
杆状病毒基因表达的时序性
杆状病毒基因的表达具有时序性，据此可以将杆状 病毒的基因分为两大类，即早期基因（early genes）和晚 期基因（late genes）。早期基因是指表达发生在病毒 DNA复制之前的基因，它又可细分为极早期基因 （immediate early genes）和早滞期基因（delayed early genes），或叫β-时相基因（β-phase genes）。晚期基因 是指那些在病毒DNA复制开始或之后才表达的基因，又 可分为晚期基因（late genes）和极晚期基因（very late genes）。杆状病毒基因表达的这种时序性调节可用级联 调节模型（regulatory cascade model）进行解释，因为目 前大多数证据表明，病毒基因的时序表达是通过时序之前 的一组病毒基因的表达产物，直接地或间接地反式激活 （trans-activeate）其时序之后的一组病毒基因的转录，因 而调节主要发生在转录水平上。
４．钱晖刘继林黄诒森， 生命的化学， 2001年21卷2期，134—136．
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杆状病毒
杆状病毒杀虫剂的缺点
毒性小，见效慢 杀虫谱窄 生产困难 稳定性差 持续能力差 注册困难
杆状病毒表达系统
LacZ:: attTn7
卡那霉素
Mini-F 复制子
外源基因
病变、外源基因表达
健康细胞
杆状病毒表达系统的优点
超强启动子：杆状病毒ph和p10基因是两种超量表达的基 因，其中ph基因的表达产物在病毒感染的晚期可达到细胞 蛋白质总量的25%-50%。 杆状病毒表达系统具有蛋白质的修饰功能， 经杆状病毒 表达系统所产生的蛋白质， 比原核系统所表达的蛋白质 更能保证其生物活性，这是因为昆虫细胞具有大部分哺乳 动物细胞所具有的翻译后修饰功能，例如磷酸化、乙酰化 和胺酰化等；只是在糖基化途径上昆虫细胞与哺乳动物细 胞有所差别，目前也可以通过构建表达特殊糖基化酶的转 基因细胞系来克服这一缺点； 杆状病毒是无脊椎动物病毒， 对人畜无害， 其生产相当 安全； 可以表达多价蛋白。目前昆虫细胞/杆状病毒系统已经成 为真核外源基因表达的最常用工具之一。
BV通ቤተ መጻሕፍቲ ባይዱ内吞进入细胞
1.病毒粒子联结到宿主细胞受体 2.宿主细胞质膜的内陷 3.含有病毒粒子的内吞泡的形成 4.内吞体的酸化 5.病毒囊膜融合蛋白的活化 6.病毒囊膜与内吞体膜的融合 7.病毒核衣壳向蛋白质的释放
杆状病毒基因组
杆状病毒的基因分类
杆状病毒的结构蛋白基因
与病毒的转录和复制相关的基因
早期时相向晚期基因表达 的过渡
杆状病毒早期基因表达时相向 晚期基因表达时相过渡的特征是病 毒DNA的复制，病毒诱导的RNA聚 合酶的活化及宿主转录的明显抑制。
晚期基因的表达
杆状病毒感染晚期时相最为显著 的特征是晚期基因的转录。晚期基因 产物分为两类
病毒粒子结构蛋白 病毒释放相关蛋白
杆状病毒分子生物学研究的 实践意义
杆状病毒的分类
NPV Nucleopholyhedrovirus 核型多角体病毒属
MNPV SNPV
Baculoviridae 杆状病毒科
GV Granulovirus 颗粒体病毒属
组I
NPV Baculoviridae
AcMNPV OpMNPV BmMNPV
LdMNPV 组 II SeMNPV HaSNPV
病毒基因表达
早期基因的表达 早期时相向晚期基因表达的过渡 晚期基因的表达
早期基因的表达
早期基因的转录是由宿主RNA聚合酶Ⅱ 介导的。早期基因迅速表达以满足病毒复制 周期所需条件：
控制宿主细胞的代谢机器 为病毒DNA复制提供必需条件 突破或阻断宿主细胞防御机制 提供下一时相病毒基因表达所需基因产物
GV
杆状病毒的结构
出芽型病毒粒子 Budded virus (BV)
BV Specific Components GP64 (F protein) FGF P6.9 VP39 VP80 P78/83 FP25K VP91 VP1054 BV/ODV-C42 BV/ODV-E26 Vlf-1 PCNA Polyhedrin PEP Occlusion body
包涵体来源型病毒粒子 Occlusion Derived virus (ODV)
ODV Specific Components ODV-E18 ODV-E25 ODV-E35 ODV-E56 ODV-E66 P74 PIF-1 PIF-2 PIF-3
杆状病毒的感染周期
中肠
几丁质
围食膜
蛋白质
糖蛋白
杆状病毒的感染周期
杆状病毒杀虫剂的研究 杆状病毒表达系统 杆状病毒作为基因治疗的载体
杆状病毒杀虫剂的研究
杆状病毒杀虫剂作为农作物、森林害虫防治 的新型安全的生物农药，已经得到了人们的重视。 与传统的化学农药相比，杆状病毒对宿主昆虫具 有高度的病原性，对非靶生物十分安全，能够在 环境中长期存活，对人、畜及其他脊椎动物无害， 不污染环境，能和其他化学农药混合使用，同时 具有防效时间长、使用方便等优点，所以早在 1973年就已被联合国粮农组织和世界卫生组织推 荐作为化学农药的理想替代品。