SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein

Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

绿色荧光蛋白分裂肽基因重组乙型肝炎病毒复制子模型的建立

为构建一种重组乙型肝炎病毒(hepatitis B virus,HBV)复制子模型,使其能够在病毒感染的细胞中表达可视化报告基因蛋白,本研究删除HBV基因组核心蛋白(HBV core,HBc)编码区部分序列,构建HBV1.1-ΔHBc113复制子载体.利用内含肽(intein)介导蛋白拼接的特性,选取加强绿色荧光蛋白(...     

Rescue and preliminary application of a recombinant newcastle disease virus expressing green fluorescent protein gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.     

In vivo tracking of Campylobacter jejuni by using a novel recombinant expressing green fluorescent protein

Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b(+) Gr-1(+) lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b(+) Gr-1(-) lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b(-) CD45R(+) B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems.     

Jellyfish green fluorescent protein as a reporter for virus infections

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.     

Construction of recombinant herpes simplex virus type I expressing green fluorescent protein without loss of any viral genes

For use in various applications in research on herpes simplex virus type 1, we attempted to generate recombinant HSV-1 expressing green fluorescent protein (GFP) without any loss of viral genes. Our results were as follows. (i) A recombinant HSV-1 (YK333), in which a GFP expression cassette driven by the Egr-1 promoter was inserted into the intergenic region between UL3 and UL4, was constructed. (ii) YK333 replicated as well as wild-type HSV-1 F strain in Vero cells. (iii) As one application of the recombinant YK333 for research on HSV-1, we developed a system to detect anti-herpetic activity, termed a fluorescence-based anti-viral assay. The 50% inhibitory concentration of ganciclovir for YK333 determined using our newly developed assay was comparable to that determined using a plaque reduction assay. YK333 will be a convenient tool for herpes simplex virus research, including such applications as monitoring of viral replication in vitro and in vivo, and rapid screening of potential anti-herpetic agents.     

Regulation of acrosome formation in mice expressing green fluorescent protein as a marker

Transgenic mice expressing enhanced green fluorescent protein under acrosin promoter were used to study the role of the Golgi complex and of the cytoskeleton during early development of the acrosomic system in exactly defined stages of the seminiferous epithelial cycle during in vitro differentiation. First acrosin expression was found uniformly in the cytoplasm of stage IV pachytene spermatocytes. The steady-state level increased up to stage X pachytene spermatocytes, and in diakinetic primary spermatocytes, acrosin started to accumulate into the Golgi complex. During step 2 of spermiogenesis, several small fluorescent proacrosomic granules were seen in various parts of the Golgi complex, and they fused to a solid acrosomic system at step 3. In cultured stage I-III seminiferous tubule segments, nocodazole slowed down acrosin incorporation and increased the distance of the acrosomic system from the nucleus. Follicle stimulating hormone had an opposite effect by increasing density of the acrosomic system together with activation of the surrounding microtubule network. The observations suggest that microtubules have an important function during the early differentiation of the acrosomic system.     

Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker

Background

The purpose of this study was to compare the effects of 0.5 fraction of inspired oxygen (FiO₂) and >0.95 FiO₂ on pulmonary gas exchange, shunt fraction and oxygen delivery (DO₂) in dorsally recumbent horses during inhalant anesthesia. The use of 0.5 FiO₂ has the potential to reduce absorption atelectasis (compared to maximal FiO₂) and augment alveolar oxygen (O₂) tensions (compared to ambient air) thereby improving gas exchange and DO₂. Our hypothesis was that 0.5 FiO₂ would reduce ventilation-perfusion mismatching and increase the fraction of pulmonary blood flow that is oxygenated, thus improving arterial oxygen content and DO₂.

Results

Arterial partial pressures of O₂ were significantly higher than preanesthetic levels at all times during anesthesia in the >0.95 FiO₂ group. Arterial partial pressures of O₂ did not change from preanesthetic levels in the 0.5 FiO₂ group but were significantly lower than in the >0.95 FiO₂ group from 15 to 90 min of anesthesia. Alveolar to arterial O₂ tension difference was increased significantly in both groups during anesthesia compared to preanesthetic values. The alveolar to arterial O₂ tension difference was significantly higher at all times in the >0.95 FiO₂ group compared to the 0.5 FiO₂ group. Oxygen delivery did not change from preanesthetic values in either group during anesthesia but was significantly lower than preanesthetic values 10 min after anesthesia in the 0.5 FiO₂ group. Shunt fraction increased in both groups during anesthesia attaining statistical significance at varying times. Shunt fraction was significantly increased in both groups 10 min after anesthesia but was not different between groups. Alveolar dead space ventilation increased after 3 hr of anesthesia in both groups.

Conclusions

Reducing FiO₂ did not change alveolar dead space ventilation or shunt fraction in dorsally recumbent, mechanically ventilated horses during 3 hr of isoflurane anesthesia. Reducing FiO₂ in dorsally recumbent isoflurane anesthetized horses does not improve oxygenation or oxygen delivery.     

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein

Background

Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV.

Methods

In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP.

Results

The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R ²?=?0.816) and ELISA (R ²?=?0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R ²?=?0.994), indicating that eGFP-NT is more accurate method to quantify nAbs.

Conclusions

Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.

     

Production of biosurfactant by a genetically-modified strain ofBacillus subtilis expressing green fluorescent protein

Biosurfactant production was investigated using two strains ofBacillus subtilis, being one a reference strain (B. subtilis 1012) and the other a genetically-modified strain (B. subtilis W1012) made able to produce the green fluorescent protein (GFP). A new method based on oil displacement technique was set up to measure the biosurfactant level in the medium. Although the tested microorganisms showed similar results in terms of cell growth parameters, the recombinant strain, besides expressing GFP, exhibited an average yield of extracellular surfactant on biomass (Y _{B/X, av} =239 mg_B g_x ^?1) more than twice that of the reference strain. The ability of the genetically-modified strain to simultaneously overproduce biosurfactant and GFP even at low cell concentration makes it an interesting candidate for possible use as a biological index-finger to monitor cell viability in bioremediation and oil recovery operations.     

Purification of green fluorescent protein overexpressed by a mutant recombinant Escherichia coli

Green fluorescent protein was purified from sonicated recombinant Escherichia coli and its mutant obtained after exposure to UV light. The latter overexpresses green fluorescent protein. The two-step procedure consisted of a two-phase aqueous extraction with PEG/salt and precipitation of the proteins from PEG phase by free Zn2+. The recoveries of green fluorescent protein were 73 and 83% in the cases of recombinant E. coli and its mutant, respectively. The corresponding fold purifications were 24 and 9, respectively. In both cases, the purified protein showed a single band on SDS-PAGE corresponding to 28 kDa.     

Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogeues could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.     

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.     

Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein

The bradyzoite stage of Toxoplasma gondii is a key step in the parasite life cycle. For a better understanding of this stage, a sensitive system to detect the tissue cysts would be required. In this study, we generated the T. gondii cyst-forming strain PLK expressing green fluorescent protein (GFP) under control of the dense granule protein 1 promoter, which works at both the tachyzoite and the bradyzoite stages. The bradyzoites with GFP fluorescence within both small and large cysts were detectable in the brain of mice infected with the recombinant PLK. Indeed, the bradyzoites expressing GFP had infectivity to mice. This study shows that transfection of the cyst-forming strain with GFP gene under control of the GRA1 promoter could be a useful approach for the study of the bradyzoite stage of T. gondii.     

Chloroplast tubules visualized in transplastomic plants expressing green fluorescent protein

A fusion between the plastid psbA promoter and the green fluorescent protein gene (gfp) was introduced into the tobacco chloroplast genome by stable plastid transformation. GFP was synthesized actively and exclusively in the chloroplasts. Tubular projections filled with GFP but containing no chlorophyll were visualized for the first time in chloroplasts of these transplastomic plants. Occasionally, the tubules connect chloroplasts with each other, suggesting the possibility of the exchange of endogenous proteins. However, the fusion of protoplasts between the transplastomic and wild-type plants showed that such chloroplast connections might be rare in mesophyll protoplasts.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Expression of recombinant green fluorescent protein in Bacillus methanolicus

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein.