为了提高猪圆环病毒2型(PCV2)衣壳(Cap)蛋白病毒样颗粒(VLPs)的产量,本试验探索了采用加热法促进杆状病毒-昆虫细胞表达的Cap蛋白体外组装的可行性,通过高效液相尺寸排阻色谱法定量PCV2 Cap VLPs,分析4~56℃加热0~10 h对细胞培养液上清...为了提高猪圆环病毒2型(PCV2)衣壳(Cap)蛋白病毒样颗粒(VLPs)的产量,本试验探索了采用加热法促进杆状病毒-昆虫细胞表达的Cap蛋白体外组装的可行性,通过高效液相尺寸排阻色谱法定量PCV2 Cap VLPs,分析4~56℃加热0~10 h对细胞培养液上清中PCV2 Cap VLPs浓度的影响,并采用透射电子显微镜(TEM)验证;通过监测45℃加热6 h后的细胞培养液上清在4℃放置6个月中PCV2 Cap VLPs的浓度变化,验证PCV2 Cap VLPs的稳定性;通过全能核酸酶消化试验验证宿主核酸对PCV2 Cap VLPs组装的作用;对比加热与未加热工艺的PCV2 Cap VLPs纯化收率,并通过高效液相尺寸排阻色谱偶联多角度激光散射、圆二色光谱、差示扫描量热法和微量热泳动分析2种工艺所得PCV2 Cap VLPs表征的一致性。结果显示,细胞培养液上清中PCV2 Cap VLPs浓度随着温度和时间的延长逐渐升高,其中45℃加热6 h后PCV2 Cap VLPs浓度为未加热的3.77倍,TEM观察进一步验证PCV2 Cap VLPs浓度增加。加热后形成的PCV2 Cap VLPs在4℃储存6个月浓度稳定,未出现解聚现象。全能核酸酶消化试验显示,加热过程中宿主核酸的作用并非关键因素。采用45℃加热6 h后再进行纯化,PCV2 Cap VLPs纯化收率是未加热工艺的2.9倍。经分析,加热与未加热工艺所得PCV2 Cap VLPs具有一致的分子量(2.385×10^(6)和2.361×10^(6) Da)、水力学半径(10.1和10.2 nm)、热转变温度Tm(67.22和66.92℃)、二级结构和特异性抗体亲和力(49.0和76.5 pmol/L),表明两者具有相近结构。本试验为PCV2 Cap VLPs的生产探索了一个提高产量的新方法,为兽用疫苗的研发提供了在抗原性质分析方法上的借鉴。展开更多
AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. ...AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.展开更多
病毒样颗粒(virus like particles,VLPs)近年来被广泛应用于疫苗和治疗性药物递送系统领域。与体内组装VLPs相比,体外组装VLPs具有组装寡聚体成分明确、反应条件可控、避免宿主成分的引入以及能实现药物活性成分的包裹和递送功能等诸多...病毒样颗粒(virus like particles,VLPs)近年来被广泛应用于疫苗和治疗性药物递送系统领域。与体内组装VLPs相比,体外组装VLPs具有组装寡聚体成分明确、反应条件可控、避免宿主成分的引入以及能实现药物活性成分的包裹和递送功能等诸多优势。概述了体外VLPs的生产方法、影响体外VLPs形成的因素、VLPs的特征分析方法,综述了体外VLPs在疫苗和治疗性药物递送方面的应用进展,期望对体外VLPs组装技术的发展提供参考思路,促进体外VLPs组装技术在疾病预防和治疗应用方面发挥更大的作用。展开更多
Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.H...Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.展开更多
文摘为了提高猪圆环病毒2型(PCV2)衣壳(Cap)蛋白病毒样颗粒(VLPs)的产量,本试验探索了采用加热法促进杆状病毒-昆虫细胞表达的Cap蛋白体外组装的可行性,通过高效液相尺寸排阻色谱法定量PCV2 Cap VLPs,分析4~56℃加热0~10 h对细胞培养液上清中PCV2 Cap VLPs浓度的影响,并采用透射电子显微镜(TEM)验证;通过监测45℃加热6 h后的细胞培养液上清在4℃放置6个月中PCV2 Cap VLPs的浓度变化,验证PCV2 Cap VLPs的稳定性;通过全能核酸酶消化试验验证宿主核酸对PCV2 Cap VLPs组装的作用;对比加热与未加热工艺的PCV2 Cap VLPs纯化收率,并通过高效液相尺寸排阻色谱偶联多角度激光散射、圆二色光谱、差示扫描量热法和微量热泳动分析2种工艺所得PCV2 Cap VLPs表征的一致性。结果显示,细胞培养液上清中PCV2 Cap VLPs浓度随着温度和时间的延长逐渐升高,其中45℃加热6 h后PCV2 Cap VLPs浓度为未加热的3.77倍,TEM观察进一步验证PCV2 Cap VLPs浓度增加。加热后形成的PCV2 Cap VLPs在4℃储存6个月浓度稳定,未出现解聚现象。全能核酸酶消化试验显示,加热过程中宿主核酸的作用并非关键因素。采用45℃加热6 h后再进行纯化,PCV2 Cap VLPs纯化收率是未加热工艺的2.9倍。经分析,加热与未加热工艺所得PCV2 Cap VLPs具有一致的分子量(2.385×10^(6)和2.361×10^(6) Da)、水力学半径(10.1和10.2 nm)、热转变温度Tm(67.22和66.92℃)、二级结构和特异性抗体亲和力(49.0和76.5 pmol/L),表明两者具有相近结构。本试验为PCV2 Cap VLPs的生产探索了一个提高产量的新方法,为兽用疫苗的研发提供了在抗原性质分析方法上的借鉴。
基金Supported by the National Science Council,No.93-2214-E-007-016 Ministry of Economic Affairs,No.93-EC-17A-17S1-0009
文摘AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
文摘病毒样颗粒(virus like particles,VLPs)近年来被广泛应用于疫苗和治疗性药物递送系统领域。与体内组装VLPs相比,体外组装VLPs具有组装寡聚体成分明确、反应条件可控、避免宿主成分的引入以及能实现药物活性成分的包裹和递送功能等诸多优势。概述了体外VLPs的生产方法、影响体外VLPs形成的因素、VLPs的特征分析方法,综述了体外VLPs在疫苗和治疗性药物递送方面的应用进展,期望对体外VLPs组装技术的发展提供参考思路,促进体外VLPs组装技术在疾病预防和治疗应用方面发挥更大的作用。
基金supported by the Special Research Fund of Ghent University and Research Foundation Flanders(FWO).CNTT is recipient of a senior postdoctoral fellowship from FWO(grant number 12V5722N).
文摘Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.
