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Peng-wei Xu Xuan Wu Hong-ning Wang Bing-cun Ma Meng-die Ding Xin Yang 《Biotechnology letters》2016,38(2):299-304
Objective
To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens.Results
IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation.Conclusions
This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses.3.
Dawei Jiang Yunchao Liu Aiping Wang Gaiping Zhang Guoyu Yang Yumei Chen Pengchao Ji Chang Liu Yapeng Song Yunfang Su Guoqiang Wang Jucai Wang Baolei Zhao Ruiguang Deng 《Biotechnology letters》2016,38(6):901-908
Objectives
To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.Results
Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.Conclusion
All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.4.
Background
Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype ? (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains.Methods
The recombinant virus PRV TK?/gE?/PrM-E+ which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK?/gE?/LacZ+ digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK?/gE?/PrM-E+ was further evaluated in mouse model.Results
A recombinant pseudorabies virus (PRV TK?/gE?/PrM-E+) was successfully constructed. Mice experiments showed that PRV TK?/gE?/PrM-E+ could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1?×?107 PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK?/gE?/PrM-E+ vaccinated mice.Conclusions
PRV TK?/gE?/PrM-E+ is a potential vaccine candidate against PRV and JEV GI infection in the future.5.
Background
Viral envelope proteins are always proposed to exert important function during virus infection and replication. Vertebrate iridoviruses are enveloped large DNA virus, which can cause great economic losses in aquaculture and ecological destruction. Although numerous iridovirus envelope proteins have been identified using bioinformatics and proteomic methods, their roles in virus infection remained largely unknown.Methods
Using SMART and TMHMM programs, we investigated the structural characteristics of Singapore grouper iridovirus (SGIV) VP19. A specific antibody against VP19 was generated and the expression profile of VP19 was clarified. The subcellular localization of VP19 in the absence or presence of other viral products was determined via transfection and immune fluorescence assay. In addition, Western blot assay and electron microscopy examination were performed to demonstrate whether SGIV VP19 was an envelope protein or a capsid protein.Results
Here, SGIV VP19 was cloned and characterized. Among all sequenced iridoviruses, VP19 and its orthologues shared common features, including 19 invariant cysteines, a proline-rich motif and a predicted transmembrane domain. Subsequently, the protein synthesis of VP19 was only detected at the late stage of SGIV infection and inhibited obviously by treating with AraC, confirming that VP19 was a late expressed protein. Ectopic expression of EGFP-VP19 in vitro displayed a punctate pattern in the cytoplasm. In SGIV infected cells, the newly synthesized VP19 protein was initially localized in the cytoplasm in a punctate pattern, and then aggregated into the virus assembly site at the late stage of SGIV infection, suggesting that other viral protein products were essential for VP19’s function during SGIV infection. In addition, Western blot assay and electron microscopy observation revealed that SGIV VP19 was associated with viral envelope, which was different from major capsid protein (MCP).Conclusion
Taken together, the current data suggested that VP19 represented a conserved envelope protein in iridovirus, and might contribute greatly to virus assembly during virus infection.6.
Meng-Shiou?Lee You-Cheng?Hseu Guan-Hua?Lai Wen-Te?Chang Hsi-Jien?Chen Chi-Hung?Huang Meng-Shiunn?Lee Min-Ying?Wang Jung-Yie?Kao Bang-Jau?You Wen-?Hsin?Lin Yi-Yang?Lien
Background
Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.Results
Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.Conclusions
Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.7.
Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector 总被引:1,自引:0,他引:1
Objective
To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.Results
The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.Conclusions
The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.8.
Background
Images of frozen hydrated [vitrified] virus particles were taken close-to-focus in an electron microscope containing structural signals at high spatial frequencies. These images had very low contrast due to the high levels of noise present in the image. The low contrast made particle selection, classification and orientation determination very difficult. The final purpose of the classification is to improve the signal-to-noise ratio of the particle representing the class, which is usually the average. In this paper, the proposed method is based on wavelet filtering and multi-resolution processing for the classification and reconstruction of this very noisy data. A multivariate statistical analysis (MSA) is used for this classification.Results
The MSA classification method is noise dependant. A set of 2600 projections from a 3D map of a herpes simplex virus -to which noise was added- was classified by MSA. The classification shows the power of wavelet filtering in enhancing the quality of class averages (used in 3D reconstruction) compared to Fourier band pass filtering. A 3D reconstruction of a recombinant virus (VP5-VP19C) is presented as an application of multi-resolution processing for classification and reconstruction.Conclusion
The wavelet filtering and multi-resolution processing method proposed in this paper offers a new way for processing very noisy images obtained from electron cryo-microscopes. The multi-resolution and filtering improves the speed and accuracy of classification, which is vital for the 3D reconstruction of biological objects. The VP5-VP19C recombinant virus reconstruction presented here is an example, which demonstrates the power of this method. Without this processing, it is not possible to get the correct 3D map of this virus.9.
Dorothea Lesche Roland Geyer Daniel Lienhard Christos T. Nakas Gaëlle Diserens Peter Vermathen Alexander B. Leichtle 《Metabolomics : Official journal of the Metabolomic Society》2016,12(10):159
Background
Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.Aim
Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.Methods
Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.Results
Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.Conclusion
Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.10.
Objective
To determine whether the G–H loop of foot-and-mouth disease virus (FMDV) serotype O can function as a target structure to harbour and display serotype Asia1 antigenic epitope at the surface.Results
Using reverse genetics, FMDV serotype O IND R2/1975 displaying a FMDV serotype Asia1 B cell epitope at the capsid surface was constructed. The epitope-inserted recombinant chimeric virus was genetically stable up to ten serial passages in cell culture and exhibited growth properties similar to the parental serotype O virus. Furthermore, the surface-displayed Asia1 epitope able to react with serotype Asia1 specific antibodies in a competitive ELISA. Importantly, the recombinant chimeric virus showed neutralizing activity to both serotype O and Asia1 polyclonal antibodies.Conclusion
The capsid protein of FMDV serotype O can effectively display potent epitope of other serotypes, making this an attractive approach for the design of new generation bi-valent FMD vaccines.11.
Yanwei Zhong Jiong Cai Chuanfu Zhang Xiaoyan Xing Enqiang Qin Jing He Panyong Mao Jun Cheng Kun Liu Dongping Xu Hongbin Song 《Virology journal》2011,8(1):542
Background
The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.Methods
The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).Results
Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.Conclusions
Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.12.
Zhaofei Wang Qiang Fu Jianguo Cao Xiyun Deng Yue Li Qiaofeng Wang Zhaofen Zheng 《Biotechnology letters》2016,38(7):1073-1079
Objectives
To evaluate the transduction efficiency of human umbilical cord-derived, late endothelial progenitor cells late (HUCB-late EPCs) with nine recombinant adeno-associated virus (rAAV) serotypes and the ability of proliferation and migration of the cells after transduction.Results
rAAV2 and rAAV6 showed a greater ability than other serotypes to transduce late EPCs (P < 0.05). After transduction, cell proliferation ability weakened (P < 0.05), but the ability of migration to stromal cell-derived factor (SDF-1) unchanged.Conclusion
There is an advantage of choosing the optimal rAAV serotype as a gene vector to alter the biologic characteristics of late EPCs.13.
Yanping Zhou Wiktor Lisowski Yan Zhou Ng Wun Jern Kama Huang Eileen Fong 《Biotechnology letters》2017,39(10):1509-1514
Objectives
To improve its phosphate accumulating abilities for phosphate recycling from wastewater, a magnetotactic bacterium, Magnetospirillum gryphiswaldense, was genetically modified to over-express polyphosphate kinase.Results
Polyphosphate kinase was over-expressed in the bacterium. The recombinant strain accumulated ninefold more polyphosphate from synthetic wastewater compared to original wild type. The magnetic property of the recombinant M. gryphiswaldense strain was retained.Conclusions
The recombinant M. gryphiswaldense can be used for phosphate removal and recovery in bioremediation.14.
N. Cesbron A.-L. Royer Y. Guitton A. Sydor B. Le Bizec G. Dervilly-Pinel 《Metabolomics : Official journal of the Metabolomic Society》2017,13(8):99
Introduction
Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.Objectives
In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.Methods
The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.Results
A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.Conclusion
The workflow generated repeatable and informative fingerprints for robust metabolome characterization.15.
Background
Fevers of unknown origin constitute a substantial disease burden in Southeast Asia. In majority of the cases, the cause of acute febrile illness is not identified.Methods
We used MassTag PCR, a multiplex assay platform, to test for the presence of 15 viral respiratory agents from 85 patients with unexplained respiratory illness representing six disease clusters that occurred in Cambodia between 2009 and 2012.Results
We detected a virus in 37 (44%) of the cases. Human rhinovirus, the virus detected most frequently, was found in both children and adults. The viruses most frequently detected in children and adults, respectively, were respiratory syncytial virus and enterovirus 68. Sequence analysis indicated that two distinct clades of enterovirus 68 were circulating during this time period.Conclusions
This is the first report of enterovirus 68 in Cambodia and contributes to the appreciation of this virus as an important respiratory pathogen.16.
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Neha Dhami Drupad K. Trivedi Royston Goodacre David Mainwaring David P. Humphreys 《Metabolomics : Official journal of the Metabolomic Society》2018,14(10):136
Introduction
Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.Objectives
The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.Methods
To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.Results
Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.Conclusion
Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.18.
Background
Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia. CAV putative intergenotypic recombinants have been reported previously. This fact is based on the previous classification of CAV sequences into three genotypes. However, it is unknown whether intersubtype recombination occurs between the recently reported four CAV genotypes and five subtypes of genome sequences.Results
Phylogenetic analysis, together with a variety of computational recombination detection algorithms, was used to investigate CAV approximately full genomes. Statistically significant evidence of intersubtype recombination was detected in the parent-like and two putative CAV recombinant sequences. This event was shown to occur between CAV subgroup A1 and A2 sequences in the phylogenetic trees.Conclusions
We revealed that intersubtype recombination in CAV genome sequences played a role in generating genetic diversity within the natural population of CAV.19.
Xue-Gang Xu Lin Gong Tian-Ling Jiang Yuan-Hong Li Xing-Hua Gao Haishan Tian Hong-Duo Chen 《Biotechnology letters》2018,40(6):1009-1014
Objectives
To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.Results
The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.Conclusions
The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.20.
Rachel A. Spicer Christoph Steinbeck 《Metabolomics : Official journal of the Metabolomic Society》2018,14(1):16