[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucl...[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .展开更多
The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV inf...The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonal serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses. The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.展开更多
Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the ...Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a（＋） vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta（DE3） E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % （73/264）. Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-20RF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.展开更多
Hepatitis C virus (HCV) encodes a single polyprotein, which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the s...Hepatitis C virus (HCV) encodes a single polyprotein, which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the synthesis of an additional protein. Until recently, studies of HCV have been hampered by the lack of a productive cell culture system. Since the identification of HCV genome approximately 17 years ago, structural, biochemical and biological information on HCV proteins has mainly been obtained with proteins produced by heterologous expression systems. In addition, some functional studies have also been confirmed with replicon systems or with retroviral particles pseudotyped with HCV envelope glycoproteins. The data that have accumulated on HCV proteins begin to provide a framework for understanding the molecular mechanisms involved in the major steps of HCV life cycle. Moreover, the knowledge accumulated on HCV proteins is also leading to the development of antiviral drugs among which some are showing promising results in early- phase clinical trials. This review summarizes the current knowledge on the functions and biochemical features of HCV proteins.展开更多
Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and nece...Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer, more efficient and economic way. The influenza subunit and VLP vaccines, taking the advantage of recombinant DNA technologies and expression system platforms, can be produced in such an ideal way. This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA), neuraminidase antigen (NA), Matrix 2 protein (M2) and nucleocapsid protein (NP). It would help to get insight into the current stage of influenza vaccines, and suggest the future design and development of novel influenza vaccines.展开更多
基金Supported by Based on Cuttingedge technology and research Project of Henan Province(072300430060)The focus of Scientific andTechnological Project of Henan Province(072102130023)Colleges and Universities of Henan Province in Support of TechnologicalInnovation Plan~~
文摘[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .
基金National Key Technologies R&D Program of China during the 10th Five-Year Plan Period(2003BA712A08-03)The Knowledge Innovation Program of the Chinese Academy of Sciences(KSCX2-YW-N-065)+1 种基金The Foundation scientific and technological project from MOST(2007FY210700)The NSFC Grant(30860255)
文摘The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonal serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses. The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.
基金supported by the special studies for social welfare researches in institutes (2005DIB4J041)
文摘Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a（＋） vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta（DE3） E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % （73/264）. Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-20RF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.
文摘Hepatitis C virus (HCV) encodes a single polyprotein, which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the synthesis of an additional protein. Until recently, studies of HCV have been hampered by the lack of a productive cell culture system. Since the identification of HCV genome approximately 17 years ago, structural, biochemical and biological information on HCV proteins has mainly been obtained with proteins produced by heterologous expression systems. In addition, some functional studies have also been confirmed with replicon systems or with retroviral particles pseudotyped with HCV envelope glycoproteins. The data that have accumulated on HCV proteins begin to provide a framework for understanding the molecular mechanisms involved in the major steps of HCV life cycle. Moreover, the knowledge accumulated on HCV proteins is also leading to the development of antiviral drugs among which some are showing promising results in early- phase clinical trials. This review summarizes the current knowledge on the functions and biochemical features of HCV proteins.
基金The Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-EW-G-8)
文摘Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer, more efficient and economic way. The influenza subunit and VLP vaccines, taking the advantage of recombinant DNA technologies and expression system platforms, can be produced in such an ideal way. This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA), neuraminidase antigen (NA), Matrix 2 protein (M2) and nucleocapsid protein (NP). It would help to get insight into the current stage of influenza vaccines, and suggest the future design and development of novel influenza vaccines.
