Recombinant Green Fluorescent Protein Research Articles

Removal of plant endogenous proteins from tobacco leaf extract by freeze–thaw treatment for purification of recombinant proteins

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze–thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at − 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant β-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Polyethyleneimine efficiently extracts recombinant cytoplasmatic green fluorescent protein produced in Escherichia coli with high purity.

We used a polycationic polymer polyethyleneimine (PEI) to develop a method to extract recombinant proteins produced in the Escherichia coli (E. coli) cytosol. Compared to high pressure homogenization, commonly used to disrupt E. coli cells, our extraction method leads to higher purity of extracts. Upon addition of PEI to the cells, flocculation occurs and the recombinant protein gradually diffuses out of the PEI/cell network. While several aspects such as the E. coli strain, the cell or PEI concentration as well as the protein titer and the pH of the buffer seem to influence the extraction rate, our results show that the PEI molecule (molecular weight and structure) must be chosen appropriately for protein extraction. The method works well with resuspended cells but can also be applied directly to fermentation broths at higher PEI concentration. This extraction approach allows for effective reduction of DNA, endotoxins, and host cell proteins levels by 2 to 4 orders of magnitude, and drastically facilitate the subsequent downstream processing steps such as centrifugation and filtration.

Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture

AbstractHydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by‐products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non‐toxic, xeno‐free, and has good mechanical properties. A recombinant spider silk protein‐derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well‐suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE‐19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future.Key points• Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125.• Two order of magnitude improvement of OriR-derived psychrophilic expression systems.• Almost twenty times enhancement in Green fluorescent protein production.

Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes.

In recent years, dendritic cells (DCs)-based vaccines have been developed to combat HIV-1 infection in preclinical and clinical trials. In this study, mice bone marrow cells-derived DCs were pulsed with the recombinant Nef, heat shock protein 27 (Hsp27) and Hsp27-Nef proteins, and also green fluorescent protein (GFP) as a positive control. Then, new platforms of DCs loaded with HIV-1 Nef and Hsp27-Nef proteins (i.e., DC prime/DC boost, DNA prime/DC boost, and DC prime/protein boost) were used to evaluate immune responses in BALB/c mice. Finally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was investigated to secret cytokines in vitro. Our data indicated that the recombinant Nef (∼30kDa), Hsp27 (∼27kDa), GFP (∼27kDa), and Hsp27-Nef (∼53kDa) proteins were greatly generated in E. coli. Moreover, the modified DCs with the recombinant proteins were prepared in large scale. The results of mice immunization showed the highest levels of antibodies, cytokines, and Granzyme B in heterologous DC prime/protein boost regimen using Hsp27-Nef antigen (DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen). The levels of IFN-γ and IL-10 cytokines in splenocytes isolated from mice immunized with DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen were higher than those in other regimens after exposure to SCR virions. These findings demonstrated the importance of Hsp27 as an adjuvant and heterologous DC prime/ protein boost regimen in improvement of immune responses. Indeed, DC Hsp27-Nef prime/ protein Hsp27-Nef boost regimen can be utilized as a promising candidate for HIV-1 vaccine development.

Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome.

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1β, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Efficient extracellular production of recombinant proteins in E. coli via enhancing expression of dacA on the genome.

D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine–Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.

An embryonic cell line from the American cockroach Periplaneta americana L. (Blattaria: Blattidae) exhibits susceptibility to AcMNPV.

Although the baculovirus expression vector system (BEVS) is widely used in the production of recombinant proteins, only a few lepidopteran insect cell lines have been successfully used so far. This study aimed at evaluating the characteristics of an embryonic cell line from the American cockroach Periplaneta americana L., RIRI-PA1, and determining whether it could be used in recombinant protein expression. Wild type Autographa californica multiple nucleopolyhedrovirus (AcMNPV-wt) and green fluorescent protein (GFP)-replicating recombinant baculoviruses (AcMNPV-GFP) were used to infect RIRI-PA1 respectively, demonstrating that RIRI-PA1 cells could be infected by AcMNPV and express recombinant proteins. Within 24 h of infection with AcMNPV-GFP, the GFP expression was higher than that in Sf21 cells. Furthermore, the infection of RIRI-PA1 cells increased gradually (multiplicity of infection, 10) within 24 h, while in Sf21 cells, the infection only began to increase within 48 h. However, after exposure for 96-168 h, the virus progeny and recombinant protein production of RIRI-PA1 cells was lower than those of Sf21 cells. Western blotting revealed that RIRI-PA1 cells could express recombinant GFP, and the protein expression level positively correlated with the multiplicity of infection. In conclusion, this is the first report that a cell line from P. americana has shown susceptibility to infection from a baculovirus and likewise express recombinant protein. Although the yield of recombinant GFP was not as high as that of Sf21 cells, the results nonetheless showed that RIRI-PA1 had an infection rate advantage in the short term (within 24 h of infection), which is of great value for further development and utilization.

Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems

Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.

Intercellular transport across pit-connections in the filamentous red alga Griffithsia monilis

Intercellular nutrient and signal transduction are essential to sustaining multicellular organisms and maximizing thebenefits of multicellularity. It has long been believed that red algal intercellular transport of macromolecules is preventedby the protein-rich pit plug within pit-connections, the only physical connection between cells. Fluorescein isothiocyanate-dextran and recombinant green fluorescence protein (rGFP) of various molecular sizes were injected into vegetativecells of Griffithsia monilis using a micromanipulator, and intercellular transport of the fluorescent probes was examined.Pit-connections were found to provide intercellular transport of tracers at rates comparable to plasmodesmata in otherorganisms. The time necessary for the transport to an adjacent cell was dependent on the molecular size and the directionof the transport. Fluorescent dextran of 3 kDa was transported to adjacent cells in 1–2 h after injection and migratedto all cells of the filament within 24 h, but fluorescent dextran of 10–20 kDa took 24 h to transfer to neighboring cells. Themigration occurred faster towards adjacent reproductive cells and to apical cells than basally. Fluorescent tracers above40 kDa and rGFP was not transported to neighboring cells, but accumulated near the pit plug. Our results suggest thatpit-connections are conduit for macromolecules between neighboring cells and that these size-specific conduits allowintercellular communication between the vegetative cells of red algae.

Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaE ΔnlpI Visualized by Electron Microscopy.

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

The new live imagers MitoMM1/2 for mitochondrial visualization

Mitochondria are eukaryotic organelles that consist of outer and inner bilayer membranes with a positive potential (H+) in the intermembrane space. This organelle plays an important role in ATP production and apoptosis. To observe the mitochondria in living cells, several fluorescent dyes (such as MitoTracker® [a standard mitochondrial imager] or rhodamine 123) have been developed. However, these reagents are unstable and exhibit a wide range of emission spectra, thereby hampering double staining results. Using recombinant DNA techniques, green or red fluorescent protein (GFP or RFP)-tagged proteins are now available for multi-color labeling of mitochondria. Here, we have discussed the development of the novel mitochondrial live imagers MitoMM1/2, derivatives of ATTO565; furthermore, MitoMM1/2 are sensitive to the membrane potential, resistant to detergents, and the fluorescence of MitoMM1/2 does not overlap with green fluorescence.

Electroporation of Mouse Follicles, Oocytes and Embryos without Manipulating Zona Pellucida.

Electroporation is an effective technique of transfection, but its efficiency depends on the optimization of various parameters. In this study, a simplified and efficient method of gene manipulation was standardized through electroporation to introduce a recombinant green fluorescent protein (GFP) construct as well as RNA-inhibitors in intact mouse follicles, oocytes and early embryos, where various electroporation parameters like voltage, pulse number and pulse duration were standardized. Electroporated preantral follicles were cultured further in vitro to obtain mature oocytes and their viability was confirmed through the localization of a known oocyte maturation marker, ovastacin, which appeared to be similar to the in vivo-derived mature oocytes and thus proved the viability of the in vitro matured oocytes after electroporation. Standardized electroporation parameters, i.e., three pulses of 30 V for 1 millisecond at an interval of 10 s, were applied to manipulate the expression of mmu-miR-26a in preantral follicles through the electroporation of miR inhibitors and mimics. The TUNEL apoptosis assay confirmed the normal development of the electroporated embryos when compared to the normal embryos. Conclusively, for the first time, this study demonstrated the delivery of exogenous oligonucleotides into intact mouse follicles, oocytes and embryos without hampering their zona pellucida (ZP) and further development.

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

Rapid and precise delivery of cells in the urethral sphincter complex by a novel needle-free waterjet technology.

To investigate the therapy of stress urinary incontinence in a preclinical setting cells were injected into the urethrae of minipigs; however, cells injected by William's needle were frequently misplaced or lost; thus, we investigated if needle-free cell injections using a novel waterjet technology facilitates precise injections in the urethral sphincter complex. Porcine adipose tissue-derived stromal cells (pADSCs) were isolated from boars, expanded, labelled, and injected in the sphincter of female pigs by waterjet employing two different protocols. After incubation for 15min or 3days, the urethrae of the pigs were examined. Injected cells were visualised by imaging and fluorescence microscopy of tissue sections. DNA of injected male cells was verified by polymerase chain reaction (PCR) of the sex-determining region (SRY) gene. Cell injections by William's needle served as controls. The new waterjet technology delivered pADSCs faster and with better on-site precision than the needle injections. Bleeding during or after waterjet injection or other adverse effects, such as swelling or urinary retention, were not observed. Morphologically intact pADSCs were detected in the urethrae of all pigs treated by waterjet. SRY-PCR of chromosomal DNA and detection of recombinant green fluorescent protein verified the injection of viable cells. In contrast, three of four pigs injected by William's needle displayed no or misplaced cells. Transurethral injection of viable pADSCs by waterjet is a simple, fast, precise, and yet gentle new technology. This is the first proof-of-principle concept study providing evidence that a waterjet injects intact cells exactly in the tissue targeted in a preclinical in vivo situation. To further explore the clinical potential of the waterjet technology longer follow-up, as well as incontinence models have to be studied.

In vitro study on promoting migration ability of rat adipose derived stem cells modified by stromal cell-derived factor 1α

To explored the effect of stromal cell-derived factor 1α (SDF-1α) on promoting the migration ability of rat adipose derived stem cells (rADSCs) by constructed the rADSCs overexpression SDF-1α via adenovirus transfection. rADSCs were isolated from adipose tissue of 6-week-old SPF Sprague Dawley rats. Morphological observation, multi-directional differentiations (osteogenic, adipogenic, and chondrogenic inductions), and flow cytometry identification were performed. Transwell cell migration experiment was used to observe and screen the optimal concentration of exogenous SDF-1α to optimize the migration ability of rADSCs; the optimal multiplicity of infection (MOI) of rADSCs was screened by observing the cell status and fluorescence expression after transfection. Then the third generation of rADSCs were divided into 4 groups: group A was pure rADSCs; group B was rADSCs co-cultured with SDF-1α at the best concentration; group C was rADSCs infected with recombinant adenovirus-mediated green fluorescent protein (Adv-GFP) with the best MOI; group D was rADSCs infected with Adv-GFP-SDF-1α overexpression adenovirus with the best MOI. Cell counting kit 8 (CCK-8) and Transwell cell migration experiment were preformed to detect and compare the effect of exogenous SDF-1α and SDF-1α overexpression on the proliferation and migration ability of rADSCs. The cell morphology, multi-directional differentiations, and flow cytometry identification showed that the cultured cells were rADSCs. After screening, the optimal stimulating concentration of exogenous SDF-1α was 12.5 nmol/L; the optimal MOI of Adv-GFP adenovirus was 200; the optimal MOI of Adv-GFP-SDF-1α overexpression adenovirus was 400. CCK-8 method and Transwell cell migration experiment showed that compared with groups A and C, groups B and D could significantly improve the proliferation and migration of rADSCs ( P<0.05); the effect of group D on enhancing the migration of rADSCs was weaker than that of group B, but the effect of promoting the proliferation of rADSCs was stronger than that of group D ( P<0.05). SDF-1α overexpression modification on rADSCs can significantly promote the proliferation and migration ability, which may be a potential method to optimize the application of ADSCs in tissue regeneration and wound repair.

Establishment of a fluorescence staining method for Schistosoma japonicum miracidia

Currently the diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. Because of the overwhelming number of impurities in the stool, eggs are rarely observed. Therefore, the stool hatching method is used to observe the miracidia in the water. However, the miracidia of Schistosoma japonicum are small and difficult to detect, and missed detection is likely to occur when the infection level is low. In this study, recombinant streptococcal protein G-enhanced green fluorescent protein (rSPG-EGFP) was expressed, purified, and used as a fluorescence staining reagent for miracidia. A preliminary miracidium fluorescence staining method based on antigen and antibody bindingwas established by combining recombinant protein staining with the stool hatching method. The stool hatching method was used to collect the miracidia of S. japonicum, and Schistosoma-positive serum and the recombinant protein were mixed to assess the feasibility of fluorescence staining of miracidia. The miracidia of S. japonicum and Schistosoma turkestanicum were incubated with S. japonicum-positive serum and S. turkestanicum-positive serum, respectively, to identify miracidia species. When the fluorescence staining method was used to observe living miracidia, the miracidiawere labelled by the recombinant protein, and their motility status was not affected.

The influence of posttranscription silensing protein-suppressor P19 on the transient gfp gene expression level in aztec tobacco plants (Nicotiana rustica L.)

Aim. Genetic constructs creation for studying the influence effect of the viral posttranscriptional silencing protein suppressor p19 on transient reporter green fluorescent protein (GFP) expression and accumulation. Methods. The Golden Gate molecular cloning method was used to create the genetic constructs; the leafy tissues of the Aztec tobacco plants (Nicotiana rustica L.) were infiltrated with a suspension of Agrobacterium tumefaciens L.; the gfp gene expression level was determined by spectrofluorometric and quantitative protein (Bradford method) assays. Results. As a result of the work, the pSPV2324 genetic construct was created, which contained the reporter gene for the green fluorescent protein gfp and the gene for the synthesis of the viral posttranscriptional silencing protein suppressor p19 and its effect on the accumulation of the recombinant GFP protein was determined. A comparative analysis of the gfp gene expression level without and with the suppressor protein synthesis gene in the genetic vector showed that the fluorescence level of GFP protein in Aztec tobacco tissues was 1.3 times higher during spectrofluorimetric analysis using the p19 suppressor gene construct. Conclusions. The positive effect of the viral suppressor silencing P19 gene on the accumulation of recombinant GFP protein in tissues plants of N. rustica L. was shown for the first time. The increase in GFP protein fluorescence when using the p19 suppressor protein construct in spectrofluorimetric analysis coincides with an increase in the total concentration of total water-soluble proteins and the level fluorescence of GFP protein in their native electrophoretic separation.&#x0D; Keywords: cloning, genetic constructs, transient expression, silencing protein suppressor p19, green fluorescent protein (GFP).

Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition.

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP.IMPORTANCE Host recognition by phage-encoded receptor binding proteins (RBPs) constitutes the first step in all phage infections and the most critical determinant of host specificity. By characterizing new types of RBPs and identifying their essential chaperones, we hope to expand the repertoire of known phage-host recognition machineries. Due to their genetic plasticity, studying RBPs and their associated chaperones can shed new light onto viral evolution affecting phage-host interactions, which is essential for fields such as phage therapy or biotechnology. In addition, since marine phages constitute one of the most important reservoirs of noncharacterized genetic diversity on the planet, their genomic and functional characterization may be of paramount importance for the discovery of novel genes with potential applications.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.