Two types of virus-like particles in the bean leaf beetle,Cerotoma trifurcata

Two types of virus-like particles were observed in the cytoplasm of hemocytes of the bean leaf beetle, Cerotoma trifurcata. The polyhedral particles were 37–40 nm in diameter and were usually in a crystalline array. They were often associated with granular and laminated structures. The enveloped, spherical particles were 70–75 nm in diameter and were composed of three parts: an outer envelope, a central electron-dense core, and an electron-lucent space between the envelope and the central core. The envelope was similar in structure to the membranes of the cell organelles. These particles were also associated with granular and filamentous structures which were distinct from those associated with the nonenveloped, smaller, polyhedral particles. The nonenveloped particles were recovered in large amounts from partially purified preparations from beetles that contained the particles in thin sections and from soybean loopers, Pseudoplusia includens, which were injected with partially purified preparations from beetles.     

Nonoccluded baculovirus- and filamentous virus-like particles in the spotted cucumber beetle,diabrotica undecimpunctata (coleoptera: chrysomelid)

Nonoccluded baculovirus-and filamentous virus-like particles were found in nuclei of hemocytes or midgut cells of field-collected spotted cucumber beetles. Each type of particle was associated with a different type of virogenic stroma containing various viral components similar to those referred to as capsid, nucleocapsid, viroplasm, and viral envelope in other known baculovirus infections. Nucleocapsids of the virus which occured only in hemocytes were rod-shaped particles approximately 230 nm long and 52 nm wide and were enveloped singly by a trilaminar unit membrane. Enveloped and partly enveloped particles appeared to be released from the nucleus to the cytoplasm by budding through the nuclear envelope acquiring additional membranes. The nucleocapsids of the virus which occurred only in nuclei of midgut cells were filamentous particles with an average diameter of 25 nm and variable length up to 2 μm. Some extremely long particles were bent almost 360° near the middle, resulting in a hairpin-like configuration. The particles were always enveloped singly. No particles budding through the nuclear envelope were observed.     

Isometric virus-like particles from citrus red mites, Panonychus citri

Virus-like spherical particles found in laboratory-reared citrus red mites but not in field populations were independent of the pathogenic virus affecting this species. Three sizes of particles are present: 18-nm spheres occuring in crystalline array, and 30- and 37-nm spheres. The particles possess antigenic properties and contain RNA.     

Construction of virus-like particles exposing HIV-1 epitopes

An original method was elaborated to construct artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface. The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates. Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested.     

A型塞内卡病毒的病毒样颗粒的制备及其免疫原性分析

为了研制A型塞内卡病毒 (Senecavirus A,SVA) 的病毒样颗粒 (Virus-like particles,VLPs) 疫苗,以SVA田间流行毒株CH-FJ-2017结构蛋白基因序列为研究对象,构建了能够同时表达SVA的3种结构蛋白VP0、VP1和VP3的单个原核重组表达质粒pET28a-SVA-VP031。通过大肠杆菌Escherichia coli表达、亲和层析纯化和体外自组装,获得SVA VLPs。透射电子显微镜鉴定显示,SVA的3种结构蛋白在体外能够自组装成直径约25–30 nm的VLPs,并且动物免疫试验结果表明,该VLPs能够有效刺激豚鼠产生高水平的抗原特异性中和抗体。上述研究结果为SVA VLPs疫苗的研制奠定了基础。     

Histopathology of virus-like particles in Heliothis spp

Larvae of Heliothis spp. collected from cotton, soybean, and peanut fields in South Carolina were found to be infected with virus-like particles (VLPs). Infected larvae became pale and swollen, stopped feeding, and remained alive for 2–3 weeks. Hemolymph from these larvae was milky and contained numerous spherical bodies ranging in diameter from 2 to 10 μm. The hemolymph also contained VLPs which were oval and measured 375 × 125 nm. Infectivity tests with crude saline extracts of infected larvae demonstrated that the pathogen could be transmitted by injection but not per os. The spherical bodies contained VLPs (387 × 149 nm) surrounded by two envelopes and packed together in clusters. These VLPs were also found in fat body cells, cuticular epithelial cells, tracheal cells, and connective tissue associated with the body wall and the gut. They were not found in muscle tissue or in midgut epithelial cells. Similar VLPs have been found in Heliothis zea from Mississippi and Trichoplusia ni from California, but a positive identification of the VLPs has not been made in any of these studies. Morphologically they appear to be distinct from any other previously described insect viruses.     

人细小病毒B19病毒样颗粒的制备

采用昆虫杆状病毒表达系统,制备人细小病毒B19病毒样颗粒(VLPs)。先通过PCR方法合成细小病毒B19衣壳蛋白基因VP2,将其克隆到pFastBac1质粒,然后转化含杆状病毒穿梭载体Bacmid的E.coliDH10Bac感受态细胞,获得重组杆状病毒表达质粒Bacmid-VP2。在脂质体介导下转染Sf9昆虫细胞,包装重组杆状病毒rBac-VP2。利用rBac-VP2感染Sf9细胞表达B19VP2蛋白,通过间接免疫荧光、Western blotting等方法鉴定目的蛋白表达。采用两次超速离心的方法对表达产物进行纯化,纯化产物在透射电镜下可见直径约22nm的VLPs。本研究成功制备了人细小病毒B19的VLPs,为B19感染血清学检测方法的建立提供了参考。     

Effect of heterotrophic nanoflagellates on the loss of virus-like particles in pond water

A decrease in the abundance of virus-like particles (VLP) by heterotrophic nanoflagellates (HNF) was examined using size-fractionated water samples taken from a hypereutrophic pond in December 1999, and in March and July 2000. We recorded a considerable decrease in the abundance of VLP in the 5.0µm filtrate relative to the 0.2–0.8µm filtrates. Decrease rates of VLP were reduced in a parallel 5.0µm filtrate treated with cycloheximide. The loss rates of VLP in 5.0µm filtrate varied in each experiment, and a high rate of loss was found when the growth rate of HNF was high. These results suggested that HNF consumed the VLP and that HNF is an important factor for decreasing viral abundance in freshwater environments.     

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

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嵌合型口蹄疫病毒样颗粒构建、表达及鉴定

为提高口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)病毒样颗粒(Virus-likeparticles,VLPs)的特异性识别和递呈,为靶向疫苗研究奠定基础,利用反向PCR技术,将卵清蛋白(Ovalbumin,OVA)第257–264位氨基酸(Amino acids,aa)的短肽嵌入FMDV结构蛋白VP3第171–172位aa或第173–174位aa,通过大肠杆菌表达FMDV结构蛋白VP0、VP1和嵌合型VP3,体外组装得到嵌合OVA257-264肽的病毒样颗粒(VLPOVA)。用动态光散射、透射电镜检测VLPOVA大小和形态,免疫印迹、酶联免疫吸附试验和激光共聚焦显微镜检测短肽的嵌入情况。结果显示在VP3的第173–174位aa嵌入OVA,不影响蛋白表达和VLPs的组装且OVA位于VLPOVA的表面,VLPOVA粒径比VLPs稍大。     

戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

Enhancement of vaccine potency by fusing modified LTK63 into human papillomavirus type 16 chimeric virus-like particles

To enhance the immunogenicity of human papillomavirus 16 (HPV 16) virus-like particles (VLPs), the modified adjuvant, mLTK63, was fused to the C-terminus of HPV 16 L2 protein. Coexpression of HPV 16 L1 and L2-mLTK63 proteins in insect cells led to the efficient assembly of HPV 16 L1/L2-mLTK63 chimeric VLPs (cVLPs), which combined the antigen and adjuvant as a unit. Compared with HPV 16 L1/L2 VLPs, the HPV 16 L1/L2-mLTK63 cVLPs had similar structural biology characteristics and binding activities with the cell surface receptors and HPV 16-specific neutralizing monoclonal antibodies. Intramuscular immunization of BALB/c mice with the HPV 16 L1/L2-mLTK63 cVLPs could induce higher titers of HPV 16-specific long-lasting neutralizing serum antibodies and stronger splenocyte proliferation, Th1- and Th2-type cytokines and CD4(+) Th responses than HPV 16 L1/L2 VLPs. The results suggested that it is possible to enhance the immunogenicity of HPV VLP vaccines via a strategy of fusing effective adjuvant protein into cVLPs.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

The release of virus-like particles from recombinant Saccharomyces cerevisiae: effect of freezing and thawing on homogenization and bead milling

Recombinant cells of Saccharomyces cerevisiae, expressing virus-like particles (Ty-VLPs), can be readily disrupted in a high pressure homogenizer and show identical disruption kinetics to the untransformed host strain. When the cells are freeze/thawed before disruption, they become about four times more resistant to homogenization. This effect increases with the number of freeze/thaw cycles, but is independent of the time the cells remain frozen. The freeze/thaw effect is observed with cells harvested during both the logarithmic and stationary phase of growth, and occurs with the untransformed host strain as well as the transformed one. Freeze/thawed cells are twice as resistant to disruption in the bead mill as fresh cells. (c) 1994 John Wiley & Sons, Inc.     

新型鸭细小病毒样颗粒的制备及鉴定

【背景】鸭短喙侏儒综合征(beak atrophy and dwarfism syndrome, BADS)是由新型鸭细小病毒(novel duck Parvovirus, NDPV)感染导致雏鸭生长发育迟缓、上下喙萎缩的疾病。BADS的暴发给我国养鸭业造成了巨大的经济损失。【目的】利用大肠杆菌表达系统制备NDPV病毒样颗粒(virus-like particles, VLPs),为研制NDPV相关疫苗奠定基础。【方法】对NDPV VP2序列全长进行密码子优化、合成,连接至pColdTF表达载体,获得pColdTF-NDPV-VP2重组质粒,酶切、测序鉴定正确后将重组质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)对蛋白表达进行可溶性分析;使用凝血酶(thrombin)切除trigger factor (TF)标签,利用镍柱(Ni-NTA)亲和层析方法纯化重组蛋白;利用Western blotting对纯化后的VP2蛋白进行反应原性分析;利用透射电镜、动态光散射观察重组蛋白形态以及能否形成VLPs。【结果】构建了pColdTF-NDPV-VP2重组质粒,在大肠杆菌中主要以可溶性形式表达,融合蛋白TF-VP2大小约为115 kDa,去除TF标签经镍柱纯化后得到67 kDa的VP2蛋白;Western blotting试验表明VP2蛋白能与NDPV鸭阳性血清发生特异性结合;通过透射电镜可以观察到形状规则、直径约为20−25 nm的病毒样颗粒。【结论】利用大肠杆菌表达系统制备了NDPV VLPs,为下一步研发BADS相关亚单位疫苗及生物相关制品提供了基础。     

Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic

Cervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virus-like particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPV-16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (approximately 3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized approximately 240 mg of L1. The chloroplast-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plant-based vaccine against HPV.     

人乳头瘤病毒18型病毒样颗粒在大肠杆菌中的表达及免疫原性分析

利用大肠杆菌表达系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,经过纯化和重组装过程获得HPV18病毒样颗粒(VLPs),研究其免疫原性和诱发中和抗体生成的水平。首先,提取HPV18的基因组DNA,通过PCR扩增获得HPV18 L1基因片段,将其插入pTrxFus表达载体,在大肠杆菌中可溶性表达HPV18 L1蛋白;其次,通过硫酸铵沉淀、离子交换层析和疏水相互作用层析获得高纯度的HPV18 L1蛋白,而后透析去除预先加入的还原剂DTT,使HPV18 L1蛋白自发组装成VLPs;最后,通过动态光散射技术和透射电子显微镜鉴定HPV18 VLPs的大小和形态,利用假病毒细胞中和实验评价HPV18 VLPs在实验动物体内的免疫原性和中和抗体生成水平。结果表明,HPV18L1蛋白可以在大肠杆菌表达系统中以可溶形式表达,经过纯化的HPV18 L1蛋白可以自发组装成为半径约为29.34nm、与HPV病毒外观相似的VLP。该VLPs在小鼠体内的中和抗体半数有效剂量为0.006μg,在兔及山羊体内诱导中和抗体滴度高达107。总之,本研究利用原核表达系统可简便高效地获得具有高度免疫原性的HPV18 VLPs,为HPV18...     

含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建

利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-CP。将FMDV的内部核糖体结合位点（IRES）保守序列连接到中间载体噬菌体基因的下游,构建原核表达载体pCPES。将重组质粒pCPES转化宿主菌BL21(DE3),1 mmol/L IPTG诱导表达。蔗糖密度梯度离心纯化表达产物。透射电镜观察到直径大约26 nm的圆形病毒样颗粒。检测病毒样颗粒的稳定性并进行RT-PCR鉴定。结果表明该病毒样颗粒含口蹄疫病毒IRES RNA序列,并且稳定性良好,本研究构建的病毒样颗粒可以作为RNA病毒检测时的标准品和质控品使用。     

Cucurbitacins and predation of the spotted cucumber beetle, Diabrotica undecimpunctata howardi

Two hypotheses about the ralationship of diabroticina beetles and plants in the family Cucurbitaceae are tested: (1) evolution of sensory receptors for cucurbitacins by diabroticina beetles was in part due to the predator protection offered by ingestion of these compounds, and (2) commercial varieties of cucurbitacin-producing cucumber offer Diabrotica undecimpunctata howardi Barber chemical protection from some potential sources of natural controlSpotted cucumber beetles fed either Marketmore 70 cucumber which contains cucurbitacin-C or Marketmore 72 which totally lacks cucurbitacin were presented to four species of vertebrate predators that commonly occur in the summer and/or winter habitats of D. u. howardi: Bufo americanus, B. fowleri, Peromyscus maniculatus, Colinus virginianus. None of the four species were significantly deterred from preying on beetles that had eaten Marketmore 70 cucumber. These results do not support either of the two hypotheses. The limitations of these negative results as evidence for refutation of the first hypothesis are discussed.

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Résumé Deux hypothèses concernant les relations entre les Diabroticina et les Cucurbitaceae ont été examinées: (1) l'évolution chez les Diabroticina des récepteurs pour les Cucurbitacines est due en partie à la protection contre les prédateurs apportée par la consommation de ces produits (2) les variétés modernes de concombres produisant de la cucurbitacine fournissent à D. undecimpunctata howardi Barber une protection chimique contre quelques sources potentielles de lutte biologique.Des D. undecimpunctata howardi, ayant consommé soit des concombres Marketmore 70 contenant de la cucurbitacine C, soit des concombres Marketmore 72 qui ont totalement perdu leur cucurbitacine, ont été présentés à quatre espèces de vertébrés prédateurs, qui s'observent fréquemment dans les habitats d'été ou d'hiver de D. undecimpunctata howardi soient Bufo americanus, B. fowleri, Peromyscus maniculatus, Colinus virginianus. Aucune de ces quatre espèces n'est significativement dissuadée d'attaquer les coléoptères qui on consommé les concombres Marketmore 70. Ces résultats n'étayent aucune des deux hypothèses. La discussion porte sur les limites de ces résultats négatifs comme preuve de la réfutation de l'hypothèse 1.