博尔纳病病毒pEGFP-p24基因重组质粒的构建及鉴定

构建博尔纳病病毒pEGFP-p24基因重组表达质粒。通过PCR方法扩增获得博尔纳病痛毒p24基因的完整序列,将此片段定向克隆到pEGFP-N1载体多克隆位点区,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性。PCR及核酸序列测定证明博尔纳病病毒pEGFP-p24基因重组表达质粒构建成功。构建的重组质粒将为研究博尔纳病病毒p24基因在真核细胞中的功能和作用提供实验依据。     

Analysis of Borna Disease Virus Trafficking in Live Infected Cells by Using a Virus Encoding a Tetracysteine-Tagged P Protein

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.     

抗博尔纳病病毒核蛋白多克隆抗体的制备和鉴定

目的利用重组博尔纳病病毒核蛋白进行动物免疫,制备多克隆抗体并对其进行鉴定。方法将重组载体pET14b-p40转化至感受态大肠埃希菌I,PTG诱导融合蛋白的表达,His-tag亲和层析纯化重组核蛋白并作为抗原免疫新西兰大白兔,收集免疫后血清,制备和纯化多克隆抗体,ELISA测定抗体效价,并进行Western-blot鉴定。结果成功制备出核蛋白多克隆抗体,ELISA检测效价高达1︰256000;该抗体与原核和真核系统中表达的核蛋白均能发生特异性反应。结论成功制备了效价和特异性良好的抗重组核蛋白多克隆抗体,为博尔纳病病毒血清免疫学检测方法的建立奠定了基础。     

A Functional Role for Neutralizing Antibodies in Borna Disease: Influence on Virus Tropism outside the Central Nervous System

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.     

Reverse-genetic approaches to the study of Borna disease virus

Borna disease virus (BDV) is an enveloped virus that has a non-segmented, negative-strand RNA genome with the characteristic organization of the mononegaviruses. However, based on its unique genetic and biological features, BDV is considered to be the prototypic member of a new mononegavirus family, the Bornaviridae. BDV causes central nervous system (CNS) disease in a wide variety of mammals. This article discusses the recently developed reverse-genetics systems for BDV, and the implications for the elucidation of the molecular mechanisms underlying BDV-host interactions, including the basis of BDV persistence in the CNS and its associated diseases.     

Hot topics in adeno-associated virus as a gene transfer vector

Adeno-associated virus (AAV) is a promising viral vector in treating many kinds of hereditary diseases. The broad host range, low level of immune response, and longevity of gene expression observed with this vector have enabled the initiation of a number of clinical trials using this gene delivery system. Another potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins. However, this virus is not well characterized. To obtain high level, persistent expression of the foreign gene, some problems should be solved. In this article, we will describe the advances in some fields of recombinant AAV technology that overcome certain limitations of the vector as a gene delivery system, such as the transduction efficiency, the production, the package capacity, and elimination of immune responses, as well as the applications involving these recombinant vectors for the treatment of some diseases.     

Functional characterization of the genomic promoter of borna disease virus (BDV): implications of 3'-terminal sequence heterogeneity for BDV persistence

Borna disease virus (BDV) is an enveloped virus with a genome organization characteristic of Mononegavirales. However, based on its unique features, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. We have described the establishment of a reverse genetics system for the rescue of BDV RNA analogues, or minigenomes, that is based on the use of polymerase I/polymerase II. Using this BDV minigenome rescue system, we have examined the functional implications of the reported sequence heterogeneity found at the 5' and 3' termini of the BDV genome and also defined the minimal BDV genomic promoter within the 3'-terminal 25 nucleotides. Our results suggest that the accumulation of RNA genome species containing truncations of one to three nucleotides at their 3' termini may contribute to modulate BDV RNA replication and gene expression during long-term persistence.     

The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

博尔纳病病毒磷蛋白在PC-12细胞内的稳定表达及对其增殖的影响

【目的】建立博尔纳病病毒磷蛋白在神经源性PC-12细胞内的稳定表达体系,初步探讨博尔纳病病毒磷蛋白对PC-12细胞的生长是否有影响。【方法】培养PC-12细胞,用阳离子脂质体的方法将带有博尔纳病病毒磷蛋白基因的表达质粒转染到细胞内进行稳定表达,用荧光显微镜和RT-PCR的方法检测细胞内磷蛋白的表达,用MTT方法检测磷蛋白对细胞生长的影响。【结果】 转染细胞经培养10代后仍然表达目的蛋白,成功建立稳定表达体系。MTT检测显示博尔纳病病毒磷蛋白对PC-12细胞的生长具有明显的抑制作用,其生长明显滞后,但粘附能力增加。【结论】 通过本文建立的体系能在PC-12细胞内稳定表达博尔纳病病毒磷蛋白,该体系可用于进一步深入研究博尔纳病病毒磷蛋白的作用机制,进而为研究博尔纳病病毒持续感染中枢神经系统的机制提供基础。此外本文通过检测细胞的增殖活性发现博尔纳病病毒磷蛋白对PC-12细胞的生长具有明显的抑制作用,可能是博尔纳病病毒持续感染中枢神经系统的重要机制之一。     

Geminivirus vectors for high-level expression of foreign proteins in plant cells

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.     

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6?weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.     

Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) inhibits experimental autoimmune encephalomyelitis in dark Agouti (DA) rats

Previous studies have shown that vascular endothelial growth factor (VEGF) expression is up-regulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a model for MS, and may exacerbate the disease. However, it remains unknown whether anti-VEGF modalities could serve as a potential treatment for such central nervous system (CNS) autoimmune diseases. We constructed a recombinant adenoviral vector carrying FLAG-tagged sFlt-1(1-3) (the first three extracellular domains of Flt-1, the hVEGF receptor-1). Intramuscular transfection of the recombinant adenoviral vector suppressed VEGF-induced inflammatory cell infiltration in matrigel plugs. When given intracerebrally to EAE rats, recombinant sFlt-1(1-3) adenoviral vector significantly reduced disease severity compared to untreated rats. sFlt-1(1-3) gene transfer blocked VEGF and greatly reduced the number of cells that express VEGF and ED1-positive cells in CNS in EAE rats. This study demonstrates that sFlt-1(1-3) gene transfer into the brain ameliorates the severity of EAE by inhibiting monocyte recruitment in the CNS of dark Agouti rats.     

Borna disease virus persistence causes inhibition of glutamate uptake by feline primary cortical astrocytes

Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.     

核桃JrGRAS2基因响应热胁迫的表达及功能分析

GRAS转录因子在植物响应逆境中起重要作用。为更好的了解核桃（Juglans regia）在逆境胁迫下的适应机制,本研究从‘香玲’核桃转录组中克隆获得一条GRAS基因（命名为JrGRAS2）,对其在不同高温胁迫下的表达进行分析,并将该基因插入酵母表达载体pYES2中构建重组载体pYES2-JrGRAS2,将pYES2-JrGRAS2转入酿酒酵母（Saccharomyces cerevisiae）INVSCI,同时以转化pYES2的重组酵母作为阴性对照,在酵母表达系统中研究该基因的抗热胁迫功能。结果显示,该基因开放读码框（ORF）全长1296bp,拟推导的蛋白分子量为47405.83Da,含有氨基酸数为431,理论等电点为5.66。在热胁迫下,JrGRAS2基因被显著诱导,特别是在36℃胁迫0.5h的茎内,其表达相对于对照被上调了335.5倍。对两种酵母进行热胁迫,发现转JrGRAS2基因酵母表现出较对照更高的生存活性。表明JrGRAS2基因具有响应热胁迫的能力,且能提高酵母的抗性,JrGRAS2基因可作为核桃逆境应答的重要候选基因。     

Reconstruction and expression of the MRI-contrast protein,ferritin, with recombinant rabies vectors

Attenuated recombinant rabies vector could be an ideal system for delivery of contrast agent gene for Magnetic Resonance Imaging (MRI) because of its neurotropic nature. In this study, the gene of a biomolecular contrast agent, ferritin, was successfully cloned into two rabies virus vectors, vaccine-based pCTN and street strain-based pNH. Recombinant virus granules were obtained and proved to express ferritin by RT-PCR after transfection of CTN-ferritin and NH-ferritin vector systems in BHK-21 cells. The recovered rabies virus-rCTN-ferritin was of similar ability to rNH-ferritin, which suggests the possibility of application of this safe and effective rabies vector system in delivery of diagnostic or therapeutic genes into the brain.     

The densovirus of Periplaneta fuliginosa (PfDNV) as an insect vector for persistent foreign gene expression in vivo

An infectious clone of the Periplaneta fuliginosa densovirus (PfDNV) has been constructed and the PfDNV genome can rescue from the plasmid and replicate as the wild-type virus in nymphs of P. fuliginosa. To investigate the ability of the cloned PfDNV genome to be used as a stable and persistent expression vector, we constructed seven recombinant plasmids in which the GFP reporter gene was inserted into the genome of PfDNV. When these recombinant constructs were transfected into hosts, the GFP was expressed efficiently in every clone. Southern blot analysis revealed that recombinant plasmids had integrated into host genome. Infectious recombinant virions could be produced from plasmids in which the GFP gene was downstream of and in frame with the NS3 and NS1 coding regions. These results indicate that PfDNV genome can be used as an insect vector for the transfer and persistent expression of an exogenous gene.