Enterovirus type 71（EV71） causes severe hand-foot-and-mouth disease （HFMD） resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the E...Enterovirus type 71（EV71） causes severe hand-foot-and-mouth disease （HFMD） resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein （EV71-P1 protein） gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GSll5. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.展开更多
Objective:The aim of our study was to investigate the effect of Pin1 on the expression of MMP-2 and MMP-9 in human colorectal carcinoma SW620 cells. Methods: We constructed a eukaryotic expression vector of RNA interf...Objective:The aim of our study was to investigate the effect of Pin1 on the expression of MMP-2 and MMP-9 in human colorectal carcinoma SW620 cells. Methods: We constructed a eukaryotic expression vector of RNA interfering (shRNA) for Pin1 gene (pGenesil-1-Pin1), and then observed its expression in SW620 cells by Western blotting. The cells motility were tested by wound healing assay and Boyden chamber assay. The protein levels and activity of MMP-2 and MMP-9 were tested by Western blotting and Gelatin zymography in SW620 cells after transfected with pGenesil-1-PIN1. Results: pGenesil-1-PIN1 was successfully constructed, which was confirmed by sequencing. Silencing the Pin1 by RNAi significantly decreased the cells motility from 96.4±3.9 per field (×10 objective) to 52.7±4.4 per field (P<0.05, Student's t-test) for SW620 cells transfected with pGenesil-1-PIN1 (SW620/p-shRNA) in Boyden chamber assay, and reduced the MMP-2 and MMP-9 expressions and activity in SW620 cells. The protein relative levels of MMP-2 were 0.32±0.04 for SW620/p-shRNA, and 0.76±0.03 for SW620/p-Con; MMP-9 were 0.41±0.09 for SW620/p-shRNA, and 0.94±0.07 for SW620/p-Con (p<0.05). Conclusion: Inhibited Pin1 expression may contribute to the suppression of the invasive and metastatic capacity of colon cancer cells in vitro.展开更多
Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments...Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments,about100 bp length,were obtained.Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1(-)andthe recombinant plasmids pcDNA3.1(-)-Humanin were identified by sequencing.Results:Recombinant plasmid DNA success-fully produced a band which had the same size as that of the thimauin positive control.The sequence of recombinant plasmidsaccorded with the Humnain gene sequence.Conclusions:A eukaryotic expression plasmid of Humanin was successfully con-structed.展开更多
Truth-conditional approaches to tense and other temporal expressions have relied on both linear-time and branching-time frameworks as needed. However, in the truth-conditional enterprise generally, it seems advisable ...Truth-conditional approaches to tense and other temporal expressions have relied on both linear-time and branching-time frameworks as needed. However, in the truth-conditional enterprise generally, it seems advisable to settle on a single depiction of time. Linear time is the simpler framework, and branching time faces difficulties for which we seem to have no immediate solutions. This paper defends the applicability of linear time. The apparent branching of time results from the use of speech acts other than factual assertion. Truth in correspondence with specific conditions is relevant when making factual assertions. However, we are in no epistemic position to advance factual assertions about the future, but only suppositions and predictions. For these, the truth-conditional content is employed differently. For suppositions and predictions, we only ＂pretend＂ to reference things, to assert facts, and to accept them as ＂true.＂ There is a wide array of alternatives about what we can suppose or predict, giving the impression of branching options. Suppositions and predictions under active consideration are often marked with present tense, whereas those that are inactive （not presently under active consideration but nonetheless relevant） are often marked with past tense.展开更多
Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused...Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused by BSE prions. PrP^Sc is thought to pathogenic factor of transmissible spongiform encephalopathy （TSE）, which invariably involve a post-translational modification process of PrPc encoded by the host euchromosome PrP gene during the period it converted into the pathogenic form （PrP^Sc）, PrP is nomal cellular protein which has been found in both neuronal and nonneuronal tissues. Since the crucial infectious event in protein-transmitted diseases is an induced misfolding of prion proteins （PrP^c） catalyzed by already misfolded PrP^Sc, it is of high importance that such collisions are enhanced by two-dimensional diffusion in cell membranes is of high importance compared to three-dimensional diffusion in solution. The level of PrP mRNA in brain is higher than other tissue, but purification of PrPc from rodent has been difficult. To understand the formation of PrP^Sc, it seemed useful to develop a system for produced a large quantities of PrPc since there is no nature source of PrP^c. The pCI-neo mammalian expression vector contains the neomycin phosphtransferase gene which serves as a marker for the selection of stable transfected cells with G418. COS-7 cells constitutively express simian viruse 40 （SV40） T-antigen and support replication of expression plasmids containing the SV40 origin of replication, amplifying the introduced expression cassettes, now become important routine of expression a large number of heterologous gene products. In this paper, the authors used pCI-neo vector to construct a recombnant pCIp264 （cotains mPrP, N-signalpeptide and C-GPI anchor） plasmid to express it in the COS-7 cells and meanwhile detect the expression fusion using IN-ELISA, IN-IFA and western blot, and obtain some approximative nature PrPc.展开更多
基金Key Project of Science and Technology Department of Wuhan(zz2011-12)Key Project of the Educational Bureau of Wuhan(sz2011-13-10)+1 种基金Project of Science and Technology Department of Hubei(2010CDB04801)The State Key Laboratory program of Viral Genetic Engineering(2010KF10)
文摘Enterovirus type 71（EV71） causes severe hand-foot-and-mouth disease （HFMD） resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein （EV71-P1 protein） gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GSll5. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.
基金Supported by a grant from the Science and Technology Project of Shanxi Province,China (No.2006031087-02)
文摘Objective:The aim of our study was to investigate the effect of Pin1 on the expression of MMP-2 and MMP-9 in human colorectal carcinoma SW620 cells. Methods: We constructed a eukaryotic expression vector of RNA interfering (shRNA) for Pin1 gene (pGenesil-1-Pin1), and then observed its expression in SW620 cells by Western blotting. The cells motility were tested by wound healing assay and Boyden chamber assay. The protein levels and activity of MMP-2 and MMP-9 were tested by Western blotting and Gelatin zymography in SW620 cells after transfected with pGenesil-1-PIN1. Results: pGenesil-1-PIN1 was successfully constructed, which was confirmed by sequencing. Silencing the Pin1 by RNAi significantly decreased the cells motility from 96.4±3.9 per field (×10 objective) to 52.7±4.4 per field (P<0.05, Student's t-test) for SW620 cells transfected with pGenesil-1-PIN1 (SW620/p-shRNA) in Boyden chamber assay, and reduced the MMP-2 and MMP-9 expressions and activity in SW620 cells. The protein relative levels of MMP-2 were 0.32±0.04 for SW620/p-shRNA, and 0.76±0.03 for SW620/p-Con; MMP-9 were 0.41±0.09 for SW620/p-shRNA, and 0.94±0.07 for SW620/p-Con (p<0.05). Conclusion: Inhibited Pin1 expression may contribute to the suppression of the invasive and metastatic capacity of colon cancer cells in vitro.
文摘Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments,about100 bp length,were obtained.Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1(-)andthe recombinant plasmids pcDNA3.1(-)-Humanin were identified by sequencing.Results:Recombinant plasmid DNA success-fully produced a band which had the same size as that of the thimauin positive control.The sequence of recombinant plasmidsaccorded with the Humnain gene sequence.Conclusions:A eukaryotic expression plasmid of Humanin was successfully con-structed.
文摘Truth-conditional approaches to tense and other temporal expressions have relied on both linear-time and branching-time frameworks as needed. However, in the truth-conditional enterprise generally, it seems advisable to settle on a single depiction of time. Linear time is the simpler framework, and branching time faces difficulties for which we seem to have no immediate solutions. This paper defends the applicability of linear time. The apparent branching of time results from the use of speech acts other than factual assertion. Truth in correspondence with specific conditions is relevant when making factual assertions. However, we are in no epistemic position to advance factual assertions about the future, but only suppositions and predictions. For these, the truth-conditional content is employed differently. For suppositions and predictions, we only ＂pretend＂ to reference things, to assert facts, and to accept them as ＂true.＂ There is a wide array of alternatives about what we can suppose or predict, giving the impression of branching options. Suppositions and predictions under active consideration are often marked with present tense, whereas those that are inactive （not presently under active consideration but nonetheless relevant） are often marked with past tense.
基金Acknowledgment This work was supported by grants from the National Natural Science Foundation （30671563, 30700597） and Key Project of Science and Technology of Gansu Province （0801NKDA034） and by gifts from the Development Planning Project of Science and Technology of Lanzhou （07-2-12, 2008- l- 167）.
文摘Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused by BSE prions. PrP^Sc is thought to pathogenic factor of transmissible spongiform encephalopathy （TSE）, which invariably involve a post-translational modification process of PrPc encoded by the host euchromosome PrP gene during the period it converted into the pathogenic form （PrP^Sc）, PrP is nomal cellular protein which has been found in both neuronal and nonneuronal tissues. Since the crucial infectious event in protein-transmitted diseases is an induced misfolding of prion proteins （PrP^c） catalyzed by already misfolded PrP^Sc, it is of high importance that such collisions are enhanced by two-dimensional diffusion in cell membranes is of high importance compared to three-dimensional diffusion in solution. The level of PrP mRNA in brain is higher than other tissue, but purification of PrPc from rodent has been difficult. To understand the formation of PrP^Sc, it seemed useful to develop a system for produced a large quantities of PrPc since there is no nature source of PrP^c. The pCI-neo mammalian expression vector contains the neomycin phosphtransferase gene which serves as a marker for the selection of stable transfected cells with G418. COS-7 cells constitutively express simian viruse 40 （SV40） T-antigen and support replication of expression plasmids containing the SV40 origin of replication, amplifying the introduced expression cassettes, now become important routine of expression a large number of heterologous gene products. In this paper, the authors used pCI-neo vector to construct a recombnant pCIp264 （cotains mPrP, N-signalpeptide and C-GPI anchor） plasmid to express it in the COS-7 cells and meanwhile detect the expression fusion using IN-ELISA, IN-IFA and western blot, and obtain some approximative nature PrPc.
