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#31047674   2019/04/30 To Up

Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture.

Yellow fever (YF) is a high-lethality viral disease, endemic in tropical regions of South America and Africa, with a population of over 900 million people under risk. A highly effective attenuated vaccine, produced in embryonated eggs, has been used for about 80 years. However, egg-based production limits manufacturing capacity, and vaccine shortage led to the emergency use of a fractional dose (1/5) by the WHO in an outbreak in Africa in 2016 and by Brazilian authorities during an outbreak in 2018. In addition, rare but fatal adverse events of this vaccine have been reported since 2001. These two aspects make clear the need for the development of a new vaccine. In an effort to develop an inactivated YF vaccine, Bio-Manguinhos/FIOCRUZ started developing a new vaccine based on the production of the attenuated 17DD virus in serum-free conditions in Vero cells propagated in bioreactors, followed by chromatography-based purification and β-propiolactone inactivation. Virus purification was studied in this work. Capture was performed using an anion-exchange membrane adsorber (Sartobind® Q), resulting in a virus recovery of 80.2 ± 4.8% and a residual DNA level of 1.3 ± 1.6 ng/dose, thus in accordance with the recommendations of the WHO (<10 ng/dose). However, the level of host cell proteins (HCP) was still high for a human vaccine, so a second chromatography step was developed based on a multimodal resin (Capto™ Core 700). This step resulted in a virus recovery of 65.7 ± 4.8% and decreased HCP levels to 345 ± 25 ppm. The overall virus recovery in these chromatography steps was 52.7%. SDS-PAGE of the purified sample showed a band with molecular mass of 56 kDa, thus consistent with the virus envelope protein (E) and corresponding to 96.7% of identified proteins. A Western blot stained with an antibody against the E protein showed a single band, confirming the identity of the sample.
Tânia P Pato, Marta C O Souza, Diogo A Mattos, Elena Caride, Davis F Ferreira, Luciane P Gaspar, Marcos S Freire, Leda R Castilho

2187 related Products with: Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture.

50 1 mg1 mg1 mg10 50 50 10 100 µg10 1 mg

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#30955631   2019/03/18 To Up

Identification of pyruvate kinase 2 as a possible crab allergen and analysis of allergenic proteins in crabs consumed in Taiwan.

In Taiwan, crab is one of the main causes for food allergy. Several proteins are recognized as crustacean allergens, and tropomyosin is known to be the major one. However, sensitization patterns of Taiwanese patients to crustacean allergens remain unclear. Therefore, we analyzed the specific-IgE binding ability of crucifix crab (Charybdis feriatus) allergens by western blot using patients' sera. In particular, we found a 56 kDa protein in crucifix crab reacted with specific-IgEs in patients' sera, and we further identified the protein as a novel crab allergen pyruvate kinase 2. Additionally, little is known about tropomyosin contents in crabs consumed in Taiwan. Thus, we also quantified the levels of tropomyosin by using enzyme-linked immunosorbent assay (ELISA) among raw and cooked crab species. Our results showed tropomyosin levels varied depending on crab species. In summary, these findings improve the understanding of crustacean allergens and contribute to the clinical diagnosis of crustacean allergies.
Chia-Ching Wu, Chih-Hung Lee, Yu-Chang Tyan, Edward S Huang, Wei-Tai Yu, Hsu-Sheng Yu

2743 related Products with: Identification of pyruvate kinase 2 as a possible crab allergen and analysis of allergenic proteins in crabs consumed in Taiwan.

25mg400Tests100 μg96 assays 1mg96 samples100 units25 mg100 assays5 mg1mg

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#30593807   2018/12/27 To Up

Molecular and structural characterization of a novel Cry1D toxin from Bacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae).

The investigation of new Bacillus thuringiensis (Bt) insecticidal proteins (Cry) with specific toxicity is one of the alternative measures used for Lepidopteran pest control. In the present study, a new Cry toxin was identified from a promising Bt strain BLB250 which was previously selected for its high toxicity against Spodoptera littoralis. The corresponding gene, designated cry1D-250, was cloned. It showed an ORF of 3498bp, encoding a protein of 1165 amino acid residues with a putative molecular mass of 132kDa which was confirmed by SDS-PAGE and Western blot analyses. The corresponding toxin named Cry1D-250 showed a higher insecticidal activity towards S. littoralis than Cry1D-133 (LC of 224.4ngcm) with an LC of only 166ngcm. Besides to the 65kDa active toxin, proteolysis activation of Cry1D-133 protein with S. littoralis midgut juice generated an extra form of 56kDa, which was the result of a second cleavage. Via activation study and 3D structure analysis, novel substitutions found in the Cry1D-250 protein compared to Cry1D-133 toxin were shown to be involved in the protein stability and toxicity. Therefore, the Cry1D-250 toxin can be considered to be an effective alternative for the control of S. littoralis.
Dalel BenFarhat-Touzri, Sonia Jemli, Fatma Driss, Slim Tounsi

2255 related Products with: Molecular and structural characterization of a novel Cry1D toxin from Bacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae).

1 mL200 ug0.1ml (1.3mg/ml)0.1 mg1 mL200 ug0.2 mg100 ul1 mg1 ml100ug1 mg

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#30261252   2018/09/25 To Up

Purification and structural-functional characterization of an exopolysaccharide from Bacillus licheniformis PASS26 with in-vitro antitumor and wound healing activities.

The exopolysaccharide (EPS) synthesized by Bacillus licheniformis PASS26, a salt pan inhabiting bacteria was successfully purified. This is the first report to reveal its structural-functional properties and biological activities. The low molecular weight (56 kDa) polysaccharide was analyzed for monosaccharide composition with TLC and HPLC. The analyses confirmed the hetero-polymeric nature of EPS with 18.44% glucose, 9.89% galactose, 16.15% fructose, 27.32% mannose and 28.18% galacturonic acid. Morphological study by scanning electron microscopy demonstrated less porous flakes like structure. Elemental analysis revealed the presence of a small quantity of nitrogen, indicating a partially charged nature of the polysaccharide. The X-ray diffraction pattern and differential scanning calorimetric (DSC) observations reflected semi-crystalline nature. Thermal gravimetric analysis (TGA) and rheological studies displayed moderate thermal stability over a range of 30-350 °C and semi-viscous nature respectively. Studies on functional properties displayed concentration-dependent water soluble nature of EPS with good water (98.8%) and oil (101.7%) holding capacity. The EPS acquired moderate emulsification activity with excellent stability against all the food grade oils and hydrocarbons tested. Studies revealed interesting in-vitro anti-tumor activity and wound healing efficiency. EPS showed significant functional and biological properties for potential applications in food and pharmaceutical industries.
Prajakta Insulkar, Savita Kerkar, S S Lele

1202 related Products with: Purification and structural-functional characterization of an exopolysaccharide from Bacillus licheniformis PASS26 with in-vitro antitumor and wound healing activities.

50 mg25 mg96T100ug2.5 mg50 ug 10 mg1 ml1000 tests100ul25 mg10 mg

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#29656213   2018/03/26 To Up

Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa.

Present study was undertaken to characterize the voltage gated potassium channel (K 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 μL diluted sperm samples namely-negative control (100 μL of sperm dilution medium (SDM) containing 10 × 10 cells), vehicle control (99 μL of SDM containing 10 × 10 cells, and DMSO- 1  μL) and 4-AP treatment group (99 μL of SDM containing 10 × 10 cells, and drug 1 μL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies are warranted to unravel the mechanistic signaling pathways involved in Kv-mediated alterations in functional dynamics of spermatozoa.
Rishi Kumar Gupta, Dilip Kumar Swain, Vijay Singh, Mukul Anand, Soumen Choudhury, Sarvajeet Yadav, Atul Saxena, Satish Kumar Garg

1051 related Products with: Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa.

50μl1 Product tipe: Instrumen100 μg100 μg100 μg100 0.1 mg5mg100μg4 Membranes/Box

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#29635067   2018/04/07 To Up

Lysine 39 of IKKε of black carp is crucial for its regulation on IRF7-mediated antiviral signaling.

Interferon regulatory factor 7 (IRF7) plays a crucial role in the interferon (IFN) signaling in mammals, in which it is activated by the TBK1/IKKε complex during host antiviral innate immune response. There are few reports about the relation between IRF7 and IKKε in teleost fishes. In this study, the IRF7 homologue (bcIRF7) of black carp (Mylopharyngodon Piceus) has been cloned and characterized. The transcription of bcIRF7 gene increased in host cells in response to the stimulation of LPS, poly (I:C) and viral infection. bcIRF7 migrated around 56 KDa in immunoblot assay and was identified as a predominantly cytosolic protein by immunofluorescent staining. bcIRF7 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF7 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). IKKε of black carp (bcIKKε) was found to be recruited into host innate immune response initiated by SVCV and GCRV in the previous work; in this paper, the kinase dead mutant of bcIKKε, bcIKKε-K39A was constructed and showed no IFN-inducing activity. The data of reporter assay and plaque assay demonstrated that bcIKKε but not bcIKKε-K39A obviously enhanced bcIRF7-mediated IFN production and antiviral activity. Our data support the conclusion that bcIKKε upregulates bcIRF7-mediated antiviral signaling, which most likely depends on its kinase activity.
Jun Li, Yu Tian, Ji Liu, Chanyuan Wang, Chaoliang Feng, Hui Wu, Hao Feng

1125 related Products with: Lysine 39 of IKKε of black carp is crucial for its regulation on IRF7-mediated antiviral signaling.

1 ml0.2 mg250 ml25ug25 µg1 mg 5 G0.1ml (1mg/ml)1 LITRE100 TESTS1 g 1 G

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#29037778   2017/10/14 To Up

Orientia tsutsugamushi infection in rodents in Anhui Province of China.

We conducted an investigation of Orientia tsutsugamushi infection among rodents in non-endemic areas in Anhui Province. Fifty-six (56) rodents including 44 Apodemus agrarius and 12 Niviventer niviventer were trapped and captured in autumn in the fields of three counties in Anhui Province. DNA samples were amplified and sequenced for the 56kDa protein gene of Orientia tsutsugamushi. The overall infection rate in the rodents was 23.2%(13/56). The rate of detection of O. tsutsugamushi in Apodemus agrarius and Niviventer niviventer were 27.3% and 8.3% respectively. Moreover, we identified two genotypes (Karp and Gilliam strains) of Orientia tsutsugamushi in rodents. Our study demonstrated that Apodemus agrarius is the main host for O. tsutsugamushi pathogen and this is the first report of Karp and Gilliam strains in these non-endemic areas in Anhui Province.
Adams Latif, Bo-Yu Liu, Zhen Chen, Yong Sun, Yong-Lin Shi, Jia Zong, Jia-Jia Li, Cui-Pin Ren, Xiao-Cheng Zhang, Xiao-Ning Liu, Xue-Jie Yu, Yan Liu

1353 related Products with: Orientia tsutsugamushi infection in rodents in Anhui Province of China.

96 tests96 tests100 μg2 mg100 μg100ug Lyophilized4 Arrays/Slide100ug

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#28964900   2017/09/28 To Up

Effect of phenol on the GABAR-coupled Cl/HCO-ATPase from fish brain: An in vitro approach on the enzyme function.

Phenol (CHOH) has a toxic effect on the central nervous system of animals and humans. The Cl/HCO-ATPase from the plasma membranes of animal brains is the primary active P-type Cl-transporting system that is coupled to GABA receptor (GABAR). In this paper, we used an in vitro approach to assess the effects of phenol (1-500μM) on the functional parameters of the Cl/HCO-ATPase isolated from the fish brain. The enzyme is insensitive to phenol in the presence of Cl or HCO in the incubation medium. By contrast, in the presence of Cl/HCO, phenol inhibits (I=27μM) both the enzyme activity and its participation in ATP-dependent Cl transport through the membranes of artificial liposomes. Enriched plasma membranes and purified enzyme preparations were separated using hrCNE-PAGE. The ATPase activity in native gels was detected in the presence of phenol (100μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300kDa, in agreement with western blot analysis with antibodies against GABAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56kDa was directly phosphorylated by γ-P-ATP and dephosphorylated in the presence of phenol. The in vitro approach described in this work allowed the first demonstration that GABAR-coupled Cl/HCO-ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.
Sergey A Menzikov

2460 related Products with: Effect of phenol on the GABAR-coupled Cl/HCO-ATPase from fish brain: An in vitro approach on the enzyme function.

100ug100.00 ul100 U96 tests

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#28610928   2017/06/10 To Up

Kinetic and biophysical characterization of a lysosomal α-l-fucosidase from the fresh water mussel, Lamellidens corrianus.

Kinetic and biophysical studies have been carried out on a lysosomal α-l-fucosidase purified from the fresh water mussel, Lamellidens corrianus. The enzyme migrates as a single band in SDS-PAGE as well as native PAGE corresponding to a M of 56kDa. Mass spectrometric analysis yielded a molecular mass of 56175.1Da for the enzyme, and peptide mass fingerprinting studies showed that it shares sequence homology with other fucosidases. Zymogram analysis showed that the α-l-fucosidase hydrolyzed 4-methyl umbelliferyl α-l-fucopyranoside. The pH and temperature optima of the enzyme were found to be 5.0-6.0 and 60°C, respectively. The K, V and k values of the enzyme estimated with p-nitrophenyl fucopyranoside are 0.85mM, 27.20 mU/mL and 1.01s, respectively. The inhibition constant (K) of the enzyme towards l-Fucose is 1.09mM. CD spectral analysis has shown that the protein contains predominantly β-sheets in its secondary structure. Chemical unfolding studies indicate that α-l-fucosidase unfolds in a broad sigmoidal, cooperative unfolding transition, centered at ∼2.2M for both guanidinium chloride and guanidinium thiocyanate. The present results obtained with the L. corrianus α-l-fucosidase are expected to provide further insights into the various biological processes associated with fucosidases and help in exploiting this enzyme in therapeutic applications.
A Venugopal, C Sudheer Kumar, Nadimpalli Siva Kumar, Musti J Swamy

1972 related Products with: Kinetic and biophysical characterization of a lysosomal α-l-fucosidase from the fresh water mussel, Lamellidens corrianus.

1mg96T1 mg100ug1,000 tests25 mg1000 tests50μl10 mg

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