AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. ...AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.展开更多
AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (P...AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.展开更多
Plasma was purified in an immobilized L-asparaginase column. The predicted results are in good agreement with experimental data. It is indicated that the mathematical model is suitable for the mass transfer and react...Plasma was purified in an immobilized L-asparaginase column. The predicted results are in good agreement with experimental data. It is indicated that the mathematical model is suitable for the mass transfer and reaction of blood purification.展开更多
Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide ...Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.展开更多
AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHO...AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.展开更多
Bacteriophages or more commonly ＂phages＂ are bacterial viruses. They are ubiquitous and good indicators of bacterial contaminations since their prevalence is high in those environments where their hosts are abundant...Bacteriophages or more commonly ＂phages＂ are bacterial viruses. They are ubiquitous and good indicators of bacterial contaminations since their prevalence is high in those environments where their hosts are abundant. Phage classification is based on morphology and for this reason, even though it is considered an old technique, TEM （Transmission Electron Microscopy） still plays a key role in their characterization. In the present work, the authors focused on TEM analysis of phage ФApr-1 isolated against Lactococcuslactis （L. lactis）, implicated in industrial fermentations and of phage ФIZSAM-1, active against Listeria monocytogenes （L. monocytogenes）, isolated from the environment. For observation with TEM （EM 900T-Zeiss）, phages were harvested in liquid media and were negative stained with fosfotungstic acid 2‰. An accurate viral ultrastructure analysis by using TEM is fundamental not only in the first approach of characterization of newly isolated phages but also for providing useful information to go further to the selection process as potential bio-decontaminants.展开更多
Canagliflozin(CANA)is a sodium-glucose co-transporter 2 inhibitor.One of the important mechanisms of CANA is the inhibitory effect on the glucose uptake in the proximal tubule of the nephron,and the other mechanism ca...Canagliflozin(CANA)is a sodium-glucose co-transporter 2 inhibitor.One of the important mechanisms of CANA is the inhibitory effect on the glucose uptake in the proximal tubule of the nephron,and the other mechanism can be the reduction of inflammatory cytokine expression monocytes and macrophages.It is proved by FDA for the management of type 2 diabetes.In the present work,we summarized the publication and clinical evidence of the CANA on healthy individuals and those with related metabolic diseases,such as type 1 and 2 diabetes,obesity,or cardiovascular and kidney diseases.This drug has been reported to offer potential advantages in regulating body weight and reducing heart failure,hypoglycemia,and stroke risk in patients with type 2 diabetes.Some in vitro and animal experiments also show that this drug has good effects on cancer treatment.However,some case reports and experiments also show the side effect of CANA,such as amputation,fracture,and pancreatitis,while the mechanism is still unknown.Overall,CANA has a good effect on the management of type 2 diabetes by reducing the risk of kidney failure,cardiovascular diseases,and stroke.However,as a new drug,more clinical trials and experiments of CANA should be carried out in the future.展开更多
基金Supported by the National Science Council,No.93-2214-E-007-016 Ministry of Economic Affairs,No.93-EC-17A-17S1-0009
文摘AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
基金Supported by the Basic Research Program from Ministry of Science and Technology of China, No. G1999054105special funds for Sino-France Center for Life Science and Genome Research from Chinese Academy of Sciences and Pasteur Institute in France
文摘AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.
基金the National Natural Science Foundation of China (No. 29806006).
文摘Plasma was purified in an immobilized L-asparaginase column. The predicted results are in good agreement with experimental data. It is indicated that the mathematical model is suitable for the mass transfer and reaction of blood purification.
基金Supported by the National Natural Science Foundation of China (No. 30200294)
文摘Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.
基金Supported by National High Technology Research and Development Program of China (863 Program), No. 2001AA215171
文摘AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.
文摘Bacteriophages or more commonly ＂phages＂ are bacterial viruses. They are ubiquitous and good indicators of bacterial contaminations since their prevalence is high in those environments where their hosts are abundant. Phage classification is based on morphology and for this reason, even though it is considered an old technique, TEM （Transmission Electron Microscopy） still plays a key role in their characterization. In the present work, the authors focused on TEM analysis of phage ФApr-1 isolated against Lactococcuslactis （L. lactis）, implicated in industrial fermentations and of phage ФIZSAM-1, active against Listeria monocytogenes （L. monocytogenes）, isolated from the environment. For observation with TEM （EM 900T-Zeiss）, phages were harvested in liquid media and were negative stained with fosfotungstic acid 2‰. An accurate viral ultrastructure analysis by using TEM is fundamental not only in the first approach of characterization of newly isolated phages but also for providing useful information to go further to the selection process as potential bio-decontaminants.
基金Beijing Excellent Talents Training Assistance(Grant No.2017000082595G244)the Health and Research Bureau of Tongzhou District(Grant No.TWKY-2016-QN-01-58)。
文摘Canagliflozin(CANA)is a sodium-glucose co-transporter 2 inhibitor.One of the important mechanisms of CANA is the inhibitory effect on the glucose uptake in the proximal tubule of the nephron,and the other mechanism can be the reduction of inflammatory cytokine expression monocytes and macrophages.It is proved by FDA for the management of type 2 diabetes.In the present work,we summarized the publication and clinical evidence of the CANA on healthy individuals and those with related metabolic diseases,such as type 1 and 2 diabetes,obesity,or cardiovascular and kidney diseases.This drug has been reported to offer potential advantages in regulating body weight and reducing heart failure,hypoglycemia,and stroke risk in patients with type 2 diabetes.Some in vitro and animal experiments also show that this drug has good effects on cancer treatment.However,some case reports and experiments also show the side effect of CANA,such as amputation,fracture,and pancreatitis,while the mechanism is still unknown.Overall,CANA has a good effect on the management of type 2 diabetes by reducing the risk of kidney failure,cardiovascular diseases,and stroke.However,as a new drug,more clinical trials and experiments of CANA should be carried out in the future.
