Recombinant Green Fluorescent Protein Research Articles

Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production

Genetically engineered (GE) algae offer the promise of producing food, fuel, and other valuable products with reduced requirements for land and fresh water. While the gains in productivity measured in GE terrestrial crops are predicted to be mirrored in GE algae, the stability of phenotypes and ecological risks posed by GE algae in large-scale outdoor cultivation remain unknown. Here, we describe the first US Environmental Protection Agency (EPA)-sanctioned experiment aimed at understanding how GE algae perform in outdoor cultivation. Acutodesmus dimorphus was genetically engineered by the addition of two genes, one for enhanced fatty acid biosynthesis, and one for recombinant green fluorescence protein (GFP) expression; both the genes and their associated phenotypes were maintained during fifty days of outdoor cultivation. We also observed that while the GE algae dispersed from the cultivation ponds, colonization of the trap ponds by the GE strain declined rapidly with increasing distance from the source cultivation ponds. In contrast, many species of indigenous algae were found in every trap pond within a few days of starting the experiment. When inoculated in water from five local lakes, the GE algae's effect on biodiversity, species composition, and biomass of native algae was indiscernible from those of the wild-type (wt) progenitor algae, and neither the GE nor wt algae were able to outcompete native strains. We conclude that GE algae can be successfully cultivated outdoors while maintaining GE traits, and that for the specific GE algal strain tested here they did not outcompete or adversely impact native algae populations when grown in water taken from local lakes. This study provides an initial evaluation of GE algae in outdoor cultivation and a framework to evaluate GE algae risks associated with outdoor GE algae production.

Acoustophoresis mediated chromatography processing: Capture of proteins from cell cultures

Chromatographic purification of target biomolecules is an important downstream process step in the development of new therapeutic agents, such as antibodies. This step employs chromatographic processes, such as affinity separation utilizing Protein A, ion exchange, or mixed mode chemistries. The workflow typically involves a packed column(s) and several chromatographic steps to achieve the desired level of purity. The steps can be time-consuming and expensive. A new process will be described, employing an acoustic standing wave in a fluid chamber to partition and maintain solid phase beads in an acoustically fluidized bed format to capture, wash, and elute the target biomolecule. Purification workflow(s) will be described for the following applications: (1) capture of a monoclonal antibody by Protein A beads from a crude cell culture system, (2) capture of recombinant Green Fluorescent protein (GFP) by anion exchange from a crude cell lysate. The workflow will include wash step(s) and recovery with a specific elution conditions. This communication will clearly demonstrate that an acoustophoresis process can purify proteins without a packed chromatography column. This new approach will minimize the time and cost involved in current purification workflows.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

BackgroundWe previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5′ untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10–80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described.ResultsPolysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5′UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5′uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories.ConclusionsRegulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Expanding the genetic code of Mus musculus

Here we report the expansion of the genetic code of Mus musculus with various unnatural amino acids including Nɛ-acetyl-lysine. Stable integration of transgenes encoding an engineered Nɛ-acetyl-lysyl-tRNA synthetase (AcKRS)/tRNAPyl pair into the mouse genome enables site-specific incorporation of unnatural amino acids into a target protein in response to the amber codon. We demonstrate temporal and spatial control of protein acetylation in various organs of the transgenic mouse using a recombinant green fluorescent protein (GFPuv) as a model protein. This strategy will provide a powerful tool for systematic in vivo study of cellular proteins in the most commonly used mammalian model organism for human physiology and disease.

Intercellular translocation of molecules via plasmodesmata in the multiseriate filamentous brown alga, Halopteris congesta (Sphacelariales, Phaeophyceae).

Despite the high number of studies on the fine structure of brown algal cells, only limited information is available on the intercelluar transportation of molecules via plasmodesmata in brown algae. In this study, plasmodesmatal permeability of Halopteris congesta was examined by observing the translocation of microinjected fluorescent tracers of different molecular sizes. The tip region of H.congesta consists of a cylindrical apical cell, while the basal region is multiseriate. Fluorescein isothiocyanate-dextran (FD; 3, 10, and 20kDa) and recombinant green fluorescent protein (27kDa) were injected into the apical cell and were observed to diffuse into the neighboring cells. FD of 40kDa was detected only in the injected apical cell. The plasmodesmatal size exclusion limit was considered to be more than 20kDa and less than 40kDa. The extent of translocation of 3 and 10kDa FD from the apical to neighboring cells 2h postinjection was estimated based on the fluorescence intensity. It was suggested that the diffusing capacity of plasmodesmata varied according to molecular size. In order to examine acropetal and/or basipetal direction of molecular movement, 3 and 10kDa FD were injected into the third cell from the apical cell. Successive observations indicated that the diffusion of fluorescence in the acropetal direction took longer than that in the basipetal direction. No ultrastructural difference in plasmodesmata was noted among the cross walls.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Next generation greener leather dyeing process through recombinant green fluorescent protein

Synthetic dyes are commonly used for dyeing and printing in paper, textile and leather industries. However, some of these dyes cause health hazards and environmental constraints. Currently, development of efficient and inexpensive eco-friendly dyes is of vital importance for dyeing industries, motivating researchers to focus on less toxic and biodegradable dyes from natural sources. The objective of this work is to develop a novel cleaner dye for leather using a green fluorescent protein (GFP) by genetic engineering approach. Specifically, two different charged variants GFP and surface modified GFP (GFPR) were evaluated for the leather dyeing process. These GFP variants were cloned using gene specific primers with restriction enzymes, successfully over expressed in Escherichia coli and purified. Dyeing conditions such as concentration of protein and duration of dyeing were optimized as 5 μM and 4 h respectively. The efficiency of protein binding was estimated through spectroscopic analysis and was around 85 and 96% for GFP and GFPR respectively. The intensity of dyeing and total color difference (ΔE) values from color measurement also indicated that GFP variants can be used as a dye for leather. Further, the characteristics of leather coated with GFP variants was studied using scanning electron microscope, thermal and UV image analysis. From the results obtained, it is suggested that GFP could serve as an environment friendly dye for leather that might reduce pollution load generated by the leather industry.

In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells.

This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place. If the recombinant SUMO-specific protease SENP1-catalytic domain is added after in situ SUMOylation, GFP signals can be erased. Therefore, the in situ SUMOylation assay can be used to assess deSUMOylation enzymatic activity.

Highly efficient and easy protease-mediated protein purification.

As both research on and application of proteins are rarely focused on the resistance towards nonspecific proteases, this property remained widely unnoticed, in particular in terms of protein purification and related fields. In the present study, diverse aspects of protease-mediated protein purification (PMPP) were explored on the basis of the complementary proteases trypsin and proteinase K as well as the model proteins green fluorescent protein (GFP) from Aequorea victoria, lipase A from Candida antarctica (CAL-A), a transaminase from Aspergillus fumigatus (AspFum), quorum quenching lactonase AiiA from Bacillus sp., and an alanine dehydrogenase from Thermus thermophilus (AlaDH). While GFP and AiiA were already known to be protease resistant, the thermostable enzymes CAL-A, AspFum, and AlaDH were selected due to the documented correlation between thermostability and protease resistance. As proof of principle for PMPP, recombinant GFP remained unaffected whereas most Escherichia coli (E. coli) host proteins were degraded by trypsin. PMPP was highly advantageous compared to the widely used heat-mediated purification of commercial CAL-A. The resistance of AspFum towards trypsin was improved by rational protein design introducing point mutation R20Q. Trypsin also served as economical and efficient substitute for site-specific endopeptidases for the removal of a His-tag fused to AiiA. Moreover, proteolysis of host enzymes with interfering properties led to a strongly improved sensitivity and accuracy of the NADH assay in E. coli cell lysate for AlaDH activity measurements. Thus, PMPP is an attractive alternative to common protein purification methods and facilitates also enzyme characterization in cell lysate.

Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

GW26-e2201 Recombinant adeno-associated virus serotype 9 transfection of atherosclerosis mice: determination of the optimal expression time in vivo

To explore the optimal time point of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein (rAAV9-eGFP) expression in the aorta of atherosclerosis mice. Atherosclerosis model was established with high-fat diet in 30 ApoE-/- mice for 16 weeks. Among them, 25 mice were

Characterization of Bovine <i>NANOG</i>5’-flanking Region during Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181) bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

High-gradient magnetic separation for technical scale protein recovery using low cost magnetic nanoparticles

Downstream processing still lacks efficient and integrated separation techniques. We present a high-gradient magnetic separation (HGMS) process for the successful purification of recombinant histidine-tagged Green Fluorescent Protein (His-GFP) from an E. coli cell lysate by means of superparamagnetic iron oxide nanoparticles functionalized with a pentadentate chelate ligand. The separator is an improved rotor stator prototype with 1L chamber volume. Using 100g of carrier, a purification performance of approx. 12g His-GFP per hour could be achieved with an eluate purity of 96% and a yield of 93% for the whole process. We demonstrate how varying processing parameters enhances the final results and provide evidence of the potential of HGMS to become a real alternative to conventional downstream processes. These pilot scale experiments show that the combination of high performance nanocarriers and optimized separator design offers an attractive system for technical implementation. Almost no nanoparticle loss took place during the experiments. The demonstration of liter scale processing with nanoparticles is important because, due to their higher surface-to-volume ratio in comparison to microparticles, it is now possible to achieve higher capacities. Furthermore, the applied nanoparticles can be seen as low-cost, very stable carriers compared to common polymer microparticles.

Silver‐nanoparticle‐Incorporated composite nanofibers for potential wound‐dressing applications

ABSTRACTThe aim of this study was to develop stable and porous poly(ethylene oxide) (PEO)–polycaprolactone blended and silver nanoparticle (Ag NP) incorporated composite nanofiber scaffolds as antibacterial wound dressings. A facile approach for the in situ synthesis of Ag NPs was explored. In this synthesis method, N,N‐dimethylformamide (DMF) was used as a solvent; it also acted as reducing agent for Ag NP formation. The stabilization of Ag NPs in the fibers was accomplished by PEO, which in turn acted as a reducing agent along with DMF. The successful synthesis of crystalline Ag NPs was confirmed by various characterization techniques. Thermogravimetric analysis, wettability, and surface roughness analysis of the nanofibers were done to examine the suitability of the scaffold for wound dressing. The as‐synthesized composite nanofibers possessed good roughness, wettability, and antibacterial potential against recombinant green fluorescent proteins expressing antibiotic‐resistant Escherichia coli. Thus, the nanofiber scaffold fabricated by this approach could serve as an ideal wound dressing. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42473.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

Initial gene vector dosing for studying symptomatology of amyotrophic lateral sclerosis in non-human primates.

Most amyotrophic lateral sclerosis (ALS) research has focused on mice, but there are distinct differences in the functional neuroanatomy of the corticospinal pathway in primates vs. rodents. A non-human primate model may be more sensitive and more predictive for therapeutic efficacy. Rhesus macaques received recombinant adeno-associated virus (AAV9) encoding either the ALS-related pathological protein TDP-43 or a green fluorescent protein (GFP) control by intravenous administration. Motor function and electromyography were assessed over a nine-month expression interval followed by post-mortem analyses. Recombinant TDP-43 or GFP was stably expressed long term. Although the TDP-43 subjects did not manifest severe paralysis and atrophy, there were trends of a partial disease state in the TDP-43 subjects relative to the control. These data indicate that a higher gene vector dose will likely be necessary for more robust effects, yet augur that a relevant primate model is feasible.

Neuroanatomical and functional characterization of CRF neurons of the amygdala using a novel transgenic mouse model

The corticotropin-releasing factor (CRF)-producing neurons of the amygdala have been implicated in behavioral and physiological responses associated with fear, anxiety, stress, food intake and reward. To overcome the difficulties in identifying CRF neurons within the amygdala, a novel transgenic mouse line, in which the humanized recombinant Renilla reniformis green fluorescent protein (hrGFP) is under the control of the CRF promoter (CRF-hrGFP mice), was developed. First, the CRF-hrGFP mouse model was validated and the localization of CRF neurons within the amygdala was systematically mapped. Amygdalar hrGFP-expressing neurons were located primarily in the interstitial nucleus of the posterior limb of the anterior commissure, but also present in the central amygdala. Secondly, the marker of neuronal activation c-Fos was used to explore the response of amygdalar CRF neurons in CRF-hrGFP mice under different experimental paradigms. C-Fos induction was observed in CRF neurons of CRF-hrGFP mice exposed to an acute social defeat stress event, a fasting/refeeding paradigm or lipopolysaccharide (LPS) administration. In contrast, no c-Fos induction was detected in CRF neurons of CRF-hrGFP mice exposed to restraint stress, forced swimming test, 48-h fasting, acute high-fat diet (HFD) consumption, intermittent HFD consumption, ad libitum HFD consumption, HFD withdrawal, conditioned HFD aversion, ghrelin administration or melanocortin 4 receptor agonist administration. Thus, this study fully characterizes the distribution of amygdala CRF neurons in mice and suggests that they are involved in some, but not all, stress or food intake-related behaviors recruiting the amygdala.

Construction and identification of recombinant adenovirus carrying human TIMP-1shRNA gene.

The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.

Inhibition of Human Parainfluenza Virus Type 2 Growth in Vitro by Catechin is caused by the Inhibition of Genome and mRNA Syntheses and by the Disruption of Cytoskeleton, and that by Tannic Acid is Mainly Caused by Genome Synthesis Inhibition and the Disruption of Cytoskeleton

The antiviral activities of catechin mixture (catechin) and tannic acid against human parainfluenza virus type 2 (hPIV-2) were investigated in vitro. Catechin and tannic acid both inhibited cell fusion induced by hPIV-2 in LLCMK2 cells. However, high concentrations of them caused cell toxicity. Both catechin and tannic acid reduced the number of viruses released from the cells. Real time PCR showed that catechin almost completely inhibited virus genome RNA synthesis, and tannic acid largely inhibited it. Virus nucleoprotein (NP), fusion (F) and hemaggulutinin-neuraminidase (HN) gene syntheses were largely inhibited by catechin, and mRNA syntheses of these proteins were partly inhibited by catechin. However, tannic acid did not cause inhibition. An indirect immunofluorescence study showed that catechin partly inhibited virus NP, F and HN protein syntheses, but tannic acid did not inhibit either virus NP, F or HN protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2∆MGFP), it was found that virus entry into the cells was not inhibited by catechin or tannic acid, and that spreading of virus to the adjacent cells was not blocked by them. Catechin and tannic acid disrupted both actin microfilaments and microtubules. These results indicated that the inhibitory effect of catechin was caused by the inhibitions of both viral genome RNA and mRNA syntheses, and by the disruption of actin microfilaments and microtubules. However, inhibition by tannic acid was mainly caused by vial genome synthesis inhibition and the disruption of cytoskeleton.