Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, con...Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.展开更多
Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome repli...Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.展开更多
Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the indu...Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.展开更多
Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays a...Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.展开更多
To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commerc...To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.展开更多
The PSV?NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was injected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subs...The PSV?NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was injected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1∶5 120 in experimental group, but it couldn’t be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV?NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.展开更多
Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well a...Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P<0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P<0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P<0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genome(NS5B and NS3) also decreased HCV viral load by 85%(P< 0.05) as compared to siRNA inhibitory effects alone(70% and 64% respectively, P<0.05). Conclusions: siRNAs directed against NS3 gene significantly decreased m RNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.展开更多
基金National Basic Research Program (973) of China ( 2009CB118701)National Natural Scientific Foundation of China (30871940, 30671615)
文摘Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.
基金National Basic Research Program of China (973 Program, Grant No. 2009CB118701)National Natural Scientific Foundation of China (Grant Nos. 30671615, 30871940)Innovation project of the Chinese Academy of Sciences (Grant No.KSCX2-YW-N-021)
文摘Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.
基金Supported by the National Natural Science Foundation of China(No.30671852)the Open Research Fund Program of the State Key Laboratory of Virology of China(Nos.2010009, 2007007) the Research Fund of the Key Laboratory of Department of Education of Liaoning Province, China(No.2009S043)
文摘Avian influenza virus（AIV） nonstructural 1（NS1） gene was amplified by real-time polymerse chain reac tion（RT-PCR） and inserted into pET28a, then transformed into E. coli BL21（DE3） competent cell. With the induction of isopropyl-β-D-thiogalactoside（IPTG） and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1（NOLC1） and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.
基金Supported by Youth Fund of Hubei Academy of Agricultural Sciences(2013NKYJJ12)
文摘Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.
基金"Penelitian Unggulan Strategis Nasional 2013" under the contract number of 0400/I1/B04/SPK-WRRI/VI/2014,Ministry of Research,Technology,and Higher Education of Indonesia,for funding this work
文摘To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.
基金Supported by Foundation of American Chinese Medicine
文摘The PSV?NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was injected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1∶5 120 in experimental group, but it couldn’t be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV?NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.
文摘Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P<0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P<0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P<0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genome(NS5B and NS3) also decreased HCV viral load by 85%(P< 0.05) as compared to siRNA inhibitory effects alone(70% and 64% respectively, P<0.05). Conclusions: siRNAs directed against NS3 gene significantly decreased m RNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.
