10 research outputs found
Efficient soluble expression and purification of influenza A and B nucleoproteins in E. coli
Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization.Viral nucleoprotein (NP) is an abundant essential protein of an influenza virus that has important functional and structural roles. It participates in genomic organization, nuclear trafficking, RNA transcription, and genome replication. From the research point of view, NP is an important protein that is used in the development of new diagnostic methods and vaccination protocols. NP is a promising target for antiviral chemotherapeutic drugs as well. Successful expression of codon-optimized NP genes in E. coli has been reported. In this study, we demonstrated the efficient expression and purification of soluble NPs of influenza A and B viruses in E. coli without the codon-optimization of DNA sequences. This procedure preserves the co-translational protein folding, protein configuration and function. Obtained NPs of influenza A and B viruses were monomers and reacted well with mouse specific antibodies according to Western blot analysis. Our results show that both influenza A and influenza B virus NPs can be efficiently expressed in E. coli without codon-optimization
ΠΠΏΠΈΠ΄Π΅ΠΌΠΈΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΠΈ ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΠ΅ Ρ Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊΠΈ ΠΎΡΡΡΡΡ ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ Π² Π‘Π°Π½ΠΊΡ-ΠΠ΅ΡΠ΅ΡΠ±ΡΡΠ³Π΅ Π² ΡΠΏΠΈΠ΄Π΅ΠΌΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΡΠ΅Π·ΠΎΠ½Ρ 2017β2018 Π³Π³. ΠΈ 2018β2019 Π³Π³.
Objective: to analyze the epidemiological and clinical features of acute respiratory infections occurring during the St. Petersburg 2017β2018 and 2018β2019 epidemic seasons.Materials and methods: the study included 457 patients, treated in St. Petersburg clinics from 2017β2019, displaying symptoms of acute respiratory infection (ARI), including evaluation of their clinical histories. Pathogen types were determined by polymerase chain reaction (PCR). Data analysis was carried out using mathematical statistics methods using the Statistica 10 software package (StatSoft Inc.).Results: in this study, we examined the epidemiological and clinical features of acute respiratory infections in St. Petersburg occurring during two epidemic seasons, 2017β2018 and 2018β2019. The 2017β2018 season was characterized by a prevalence of infections caused by influenza B viruses and influenza A subtype H3N2 viruses. In the 2018β2019 season, there was a greater number of acute respiratory viral infections (ARVIs) and infections caused by influenza A subtype H1N1pdm; influenza B virus was detected only in isolated cases. In the 2017β2018 sore throats and muscle aches were a characteristic symptom of influenza A H1N1pdm infections, of bacterial infections β only sore throats. It was shown that throat pain and vasodilation of the scleral and soft palate vessels were significantly more frequent in the 2017β2018 season, compared to the 2018β2019 season. Cough and redness of the posterior pharyngeal wall were hallmark signs of ARVIs in the 2018β2019 season.Conclusion: according to the data, each epidemic season is characterized not only by its own type-specific acute respiratory infection frequencies, but also by different clinical manifestation frequencies. For global monitoring, treatment effectiveness evaluation, and refined study of acute respiratory infection clinical features, it is advisable to use approaches which incorporate accurate, specific, and rapid molecular biological methods capable of identifying a broad range of pathogens.Β Π¦Π΅Π»Ρ: ΡΡΠ°Π²Π½ΠΈΡΠ΅Π»ΡΠ½ΡΠΉ Π°Π½Π°Π»ΠΈΠ· ΡΠΏΠΈΠ΄Π΅ΠΌΠΈΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
Π΄Π°Π½Π½ΡΡ
ΠΈ ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΡ
Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊ ΡΠ΅ΡΠ΅Π½ΠΈΡ ΠΎΡΡΡΡΡ
ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ Ρ Π±ΠΎΠ»ΡΠ½ΡΡ
Π² Π‘Π°Π½ΠΊΡ-ΠΠ΅ΡΠ΅ΡΠ±ΡΡΠ³Π΅ Π² ΡΠΏΠΈΠ΄Π΅ΠΌΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΡΠ΅Π·ΠΎΠ½Ρ 2017β2018 Π³Π³. ΠΈ 2018β2019 Π³Π³.ΠΠ°ΡΠ΅ΡΠΈΠ°Π»Ρ ΠΈ ΠΌΠ΅ΡΠΎΠ΄Ρ: Π² ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ Π²ΠΊΠ»ΡΡΠ΅Π½ΠΎ 457 ΠΏΠ°ΡΠΈΠ΅Π½ΡΠΎΠ², Π½Π°Ρ
ΠΎΠ΄ΠΈΠ²ΡΠΈΡ
ΡΡ Π½Π° Π»Π΅ΡΠ΅Π½ΠΈΠΈ Π² ΠΊΠ»ΠΈΠ½ΠΈΠΊΠ°Ρ
Π‘Π°Π½ΠΊΡ-ΠΠ΅ΡΠ΅ΡΠ±ΡΡΠ³Π° Π² 2017β2019 Π³Π³., Ρ ΡΠΈΠΌΠΏΡΠΎΠΌΠ°ΠΌΠΈ ΠΎΡΡΡΡΡ
ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ, ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ Π°Π½Π°Π»ΠΈΠ· ΡΠΏΠΈΠ΄Π΅ΠΌΠΈΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
Π΄Π°Π½Π½ΡΡ
ΠΈ ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΡ
Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊ ΡΠ΅ΡΠ΅Π½ΠΈΡ Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΡ. ΠΠ΅ΡΠΎΠ΄ΠΎΠΌ ΠΏΠΎΠ»ΠΈΠΌΠ΅ΡΠ°Π·Π½ΠΎΠΉ ΡΠ΅ΠΏΠ½ΠΎΠΉ ΡΠ΅Π°ΠΊΡΠΈΠΈ ΠΎΠΏΡΠ΅Π΄Π΅Π»Π΅Π½ Π²ΠΈΠ΄ ΠΏΠ°ΡΠΎΠ³Π΅Π½Π°. Π‘ΡΠ°ΡΠΈΡΡΠΈΡΠ΅ΡΠΊΠΈΠΉ Π°Π½Π°Π»ΠΈΠ· ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠ² ΠΌΠ°ΡΠ΅ΠΌΠ°ΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΡΡΠ°ΡΠΈΡΡΠΈΠΊΠΈ ΠΏΡΠΈ ΠΏΠΎΠΌΠΎΡΠΈ ΠΏΠ°ΠΊΠ΅ΡΠ° Statistica 10, StatSoft Inc.Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ. Π ΡΠ΅Π·ΠΎΠ½ 2017β2018 Π³Π³. Π±ΡΠ»ΠΎ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠ½ΠΎ ΠΏΡΠ΅Π²Π°Π»ΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ, Π²ΡΠ·Π²Π°Π½Π½ΡΡ
Π²ΠΈΡΡΡΠ°ΠΌΠΈ Π³ΡΠΈΠΏΠΏΠ° Π ΠΈ Π (H3N2). Π ΡΠ΅Π·ΠΎΠ½Π΅ 2018β2019 Π³Π³. Π±ΡΠ»ΠΎ ΠΎΡΠΌΠ΅ΡΠ΅Π½ΠΎ Π±ΠΎΠ»ΡΡΠ΅Π΅ ΡΠΈΡΠ»ΠΎ ΡΠ»ΡΡΠ°Π΅Π² ΠΎΡΡΡΡΡ
ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ
Π²ΠΈΡΡΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ, Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ Π³ΡΠΈΠΏΠΏΠ°, Π²ΡΠ·Π²Π°Π½Π½ΠΎΠ³ΠΎ Π²ΠΈΡΡΡΠΎΠΌ Π³ΡΠΈΠΏΠΏΠ° Π (H1N1pdm); Π²ΠΈΡΡΡ Π³ΡΠΈΠΏΠΏΠ° Π ΡΠ΅Π³ΠΈΡΡΡΠΈΡΠΎΠ²Π°Π»ΠΈ Π² Π΅Π΄ΠΈΠ½ΠΈΡΠ½ΡΡ
ΡΠ»ΡΡΠ°ΡΡ
. Π₯Π°ΡΠ°ΠΊΡΠ΅ΡΠ½ΡΠΌ ΡΠΈΠΌΠΏΡΠΎΠΌΠΎΠΌ Π² ΡΠ΅Π·ΠΎΠ½Π΅ 2017β2018 Π³Π³. Π΄Π»Ρ Π³ΡΠΈΠΏΠΏΠ° Π (H1N1pdm) Π±ΡΠ»ΠΈ ΠΌΡΡΠ΅ΡΠ½ΡΠ΅ Π±ΠΎΠ»ΠΈ ΠΈ Π±ΠΎΠ»ΠΈ Π² Π³ΠΎΡΠ»Π΅, Π΄Π»Ρ Π±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ β ΡΠ°ΠΊΠΆΠ΅ Π±ΠΎΠ»ΠΈ Π² Π³ΠΎΡΠ»Π΅. ΠΠΎΠ»ΠΈ Π² Π³ΠΎΡΠ»Π΅ ΠΈ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΈ ΡΠΎΡΡΠ΄ΠΎΠ² ΡΠΊΠ»Π΅Ρ ΠΈ ΠΌΡΠ³ΠΊΠΎΠ³ΠΎ Π½Π΅Π±Π° Π΄ΠΎΡΡΠΎΠ²Π΅ΡΠ½ΠΎ ΡΠ°ΡΠ΅ Π±ΡΠ»ΠΈ ΠΎΡΠΌΠ΅ΡΠ΅Π½Ρ Π² ΡΠ΅Π·ΠΎΠ½Π΅ 2017β2018 Π³Π³. ΠΏΠΎ ΡΡΠ°Π²Π½Π΅Π½ΠΈΡ Ρ ΡΠ΅Π·ΠΎΠ½ΠΎΠΌ 2018β2019 Π³Π³. ΠΠ°ΡΠ΅Π»Ρ ΠΈ Π³ΠΈΠΏΠ΅ΡΠ΅ΠΌΠΈΡ Π·Π°Π΄Π½Π΅ΠΉ ΡΡΠ΅Π½ΠΊΠΈ Π³Π»ΠΎΡΠΊΠΈ Π±ΡΠ»ΠΈ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠ½ΡΠΌΠΈ ΡΠΈΠΌΠΏΡΠΎΠΌΠ°ΠΌΠΈ Π΄Π»Ρ ΠΠ ΠΠ Π² ΡΠ΅Π·ΠΎΠ½Π΅ 2018β2019 Π³Π³.ΠΠ°ΠΊΠ»ΡΡΠ΅Π½ΠΈΠ΅: ΡΠ°ΡΡΠΌΠΎΡΡΠ΅Π½Π½ΡΠ΅ Π΄Π²Π° ΡΠΏΠΈΠ΄Π΅ΠΌΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠ΅Π·ΠΎΠ½Π° Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΠΎΠ²Π°Π»ΠΈΡΡ Π½Π΅ ΡΠΎΠ»ΡΠΊΠΎ ΡΠ°Π·Π»ΠΈΡΠ½ΠΎΠΉ ΡΠ°ΡΡΠΎΡΠΎΠΉ ΠΎΡΡΡΡΡ
ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ, Π½ΠΎ ΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΠΎΠΉ ΡΠ°ΡΡΠΎΡΠΎΠΉ ΠΈΡ
ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΏΡΠΎΡΠ²Π»Π΅Π½ΠΈΠΉ. ΠΠ»Ρ Π³Π»ΠΎΠ±Π°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΌΠΎΠ½ΠΈΡΠΎΡΠΈΠ½Π³Π°, ΠΎΡΠ΅Π½ΠΊΠΈ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π»Π΅ΡΠ΅Π½ΠΈΡ ΠΈ Π±ΠΎΠ»Π΅Π΅ Π΄Π΅ΡΠ°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΈΠ·ΡΡΠ΅Π½ΠΈΡ ΠΊΠ»ΠΈΠ½ΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΎΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠ΅ΠΉ ΠΎΡΡΡΡΡ
ΡΠ΅ΡΠΏΠΈΡΠ°ΡΠΎΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ ΡΠ΅Π»Π΅ΡΠΎΠΎΠ±ΡΠ°Π·Π½ΠΎ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°ΡΡ ΡΠΎΡΠ½ΡΠ΅, ΡΠΏΠ΅ΡΠΈΡΠΈΡΠ½ΡΠ΅ ΠΈ Π±ΡΡΡΡΡΠ΅ ΠΌΠΎΠ»Π΅ΠΊΡΠ»ΡΡΠ½ΠΎ-Π±ΠΈΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΠΌΠ΅ΡΠΎΠ΄Ρ Ρ Π±ΠΎΠ»ΡΡΠΈΠΌ ΠΊΠΎΠ»ΠΈΡΠ΅ΡΡΠ²ΠΎΠΌ ΠΎΠΏΡΠ΅Π΄Π΅Π»ΡΠ΅ΠΌΡΡ
ΠΏΠ°ΡΠΎΠ³Π΅Π½ΠΎΠ².
APPLICATION OF SOLID-PHASE, ENZYME-LINKED IMMUNOASSAYS IN MICROARRAY FORMATE FOR QUANTITATIVE DETERMINATION OF CYTOKINES
Abstract. Present work describes a multiplex βsandvichβ approach to enzyme-linked, solid-phase immunosorbent assay designed in microarray formate, aiming for quantitative evaluation of individual profile of cytokine expression. Its high specificity, throughput and sensitivity (30 to 20,000 pg/mL) are demonstrated with human IL-2, IL-4, IL-10, and TNF assays. This diagnostic technique may be further used both for research applications and in clinical trials
Epidemiological and clinical features of acute respiratory infections occurring in St. Petersburg during the 2017β2018 and 2018β2019 epidemic seasons
Objective: to analyze the epidemiological and clinical features of acute respiratory infections occurring during the St. Petersburg 2017β2018 and 2018β2019 epidemic seasons.Materials and methods: the study included 457 patients, treated in St. Petersburg clinics from 2017β2019, displaying symptoms of acute respiratory infection (ARI), including evaluation of their clinical histories. Pathogen types were determined by polymerase chain reaction (PCR). Data analysis was carried out using mathematical statistics methods using the Statistica 10 software package (StatSoft Inc.).Results: in this study, we examined the epidemiological and clinical features of acute respiratory infections in St. Petersburg occurring during two epidemic seasons, 2017β2018 and 2018β2019. The 2017β2018 season was characterized by a prevalence of infections caused by influenza B viruses and influenza A subtype H3N2 viruses. In the 2018β2019 season, there was a greater number of acute respiratory viral infections (ARVIs) and infections caused by influenza A subtype H1N1pdm; influenza B virus was detected only in isolated cases. In the 2017β2018 sore throats and muscle aches were a characteristic symptom of influenza A H1N1pdm infections, of bacterial infections β only sore throats. It was shown that throat pain and vasodilation of the scleral and soft palate vessels were significantly more frequent in the 2017β2018 season, compared to the 2018β2019 season. Cough and redness of the posterior pharyngeal wall were hallmark signs of ARVIs in the 2018β2019 season.Conclusion: according to the data, each epidemic season is characterized not only by its own type-specific acute respiratory infection frequencies, but also by different clinical manifestation frequencies. For global monitoring, treatment effectiveness evaluation, and refined study of acute respiratory infection clinical features, it is advisable to use approaches which incorporate accurate, specific, and rapid molecular biological methods capable of identifying a broad range of pathogens
Ceruloplasmin gene expression profile changes in the rat mammary gland during pregnancy, lactation and involution
Copper metabolism disturbances in mammary gland (MG) cells have severe consequences in newborns. The mechanism that controls the balance of copper in the MG has not been thoroughly characterized. Four primary copper homeostasis genes in mammals: (1) ceruloplasmin (Cp) encoding multifunction multicopper blue (ferr)oxidase; (2) CTR1 encoding high affinity copper importer 1; and (3 and 4) two similar genes encoding Cu(I)/Cu(II)-ATPases P1 type (ATP7A and ATP7B) responsible for copper efflux from the cells and metallation of cuproenzymes formed in the Golgi complex are expressed in MG. This study aimed to characterize expression of these genes during pregnancy, lactation and forced involution in the rat MG. We found that Cp anchored to the plasma membrane and ATP7A were expressed during pregnancy and lactation. Soluble Cp and ATP7B were highly expressed in lactating MG decreasing to its ending. CTR1 activity increased during MG growth and reached its maximum at postpartum and then it decreased until the end of lactation. During early forced MG involution, Cp gene expression persisted; while a form of Cp that lacked exon 18 appeared. We suggest that Cp gene expressional changes at the transcriptional and posttranscriptional level reflect various physiological functions of Cp proteins during MG remodeling
MOESM2 of The influenza A virus NS genome segment displays lineage-specific patterns in predicted RNA secondary structure
Additional file 2. List of sequences and RNA second structure forΓΒ Γ’ΒΒNt 497Γ’ΒΒ564
Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1
Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription