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 find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion ...AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.展开更多
AIM： To evaluate the efficacy of 5 compared to ：tO granulocyteaphaeresis sessions in patients with active steroid-dependent ulcerative colitis. METHODS： In this pilot, prospective, multicenter randomized trial, 20 ...AIM： To evaluate the efficacy of 5 compared to ：tO granulocyteaphaeresis sessions in patients with active steroid-dependent ulcerative colitis. METHODS： In this pilot, prospective, multicenter randomized trial, 20 patients with moderately active steroid-dependent ulcerative colitis were randomized to 5 or 10 granulocyteaphaeresis sessions. The primary objective was clinical remission at wk 17. Secondary measures included endoscopic remission and steroid consumption.RESULTS： Nine patients were randomized to 5 granulocyteaphaeresis sessions （group 1） and 11 patients to 10 granulocyteaphaeresis sessions （group 2）. At wk 17, 37.5% of patients in group 1 and 45.45% of patients in group 2 were in clinical remission. Clinical remission was accompanied by endoscopic remission in all cases. Eighty-six percent of patients achieving remission were steroid-free at wk 17. Daily steroid requirements were significantly lower in group 2. Eighty-nine per cent of patients remained in remission during a one year follow-up. One serious adverse event, not related to the study therapy, was reported. CONCLUSION： Granulocyteaphaeresis is safe and effective for the treatment of steroid-dependent ulcerative colitis. In this population, increasing the number of aphaeresis sessions is not associated with higher remission rates, but affords a significant steroid-sparing effect.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
To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacter...To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.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.
文摘AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.
文摘AIM： To evaluate the efficacy of 5 compared to ：tO granulocyteaphaeresis sessions in patients with active steroid-dependent ulcerative colitis. METHODS： In this pilot, prospective, multicenter randomized trial, 20 patients with moderately active steroid-dependent ulcerative colitis were randomized to 5 or 10 granulocyteaphaeresis sessions. The primary objective was clinical remission at wk 17. Secondary measures included endoscopic remission and steroid consumption.RESULTS： Nine patients were randomized to 5 granulocyteaphaeresis sessions （group 1） and 11 patients to 10 granulocyteaphaeresis sessions （group 2）. At wk 17, 37.5% of patients in group 1 and 45.45% of patients in group 2 were in clinical remission. Clinical remission was accompanied by endoscopic remission in all cases. Eighty-six percent of patients achieving remission were steroid-free at wk 17. Daily steroid requirements were significantly lower in group 2. Eighty-nine per cent of patients remained in remission during a one year follow-up. One serious adverse event, not related to the study therapy, was reported. CONCLUSION： Granulocyteaphaeresis is safe and effective for the treatment of steroid-dependent ulcerative colitis. In this population, increasing the number of aphaeresis sessions is not associated with higher remission rates, but affords a significant steroid-sparing effect.
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.
文摘To purify and identify the simian parvovirus （SPV） protein Vp2 expressed in E. coli, fusion protein of SPV Vp2 was expressed in E. coli DHSα competent cells transformed with vector pThioHis AVp2, and the new bacterial protein extraction reagent was used to extract the protein. Detergents with different characteristics were used to solubilize the fusion protein, and metal chelating resin （ProBond） with a continuous elusion polyacrylamide gel electrophoresis procedure was employed to purify the fusion protein. SDS-PAGE gel stained with coomassie blue and Western-blotting probed with anti-thio and anti-SPV Vp2 antibodies were used to identify the specificity of the expressed and purified fusion proteins. It was found that the SPV Vp2 protein expressed in E. coli was highly insoluble, and could not be solublized by the commonly used detergent. However, 6 M urea could solubilize the fusion proteins and was then employed for the further purification procedure, but metal chelating resin could not be used for this procedure, because of the loss of the tertiary structure of HP-thiaoredoxin and the metal-binding domain. The technique with continuous elusion polyacrylamide gel electrophoresis yielded a homogenous protein with a single band on the gel stained with coomassie blue and retained reactivity with anti-thio or anti-SPV Vp2 antibodies. It is evident that this technique with successful purification of SPV Vp2 protein has practical significance for the further investigation on the simian parvovirus infection.
