A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a reco...A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.展开更多
It has now believed that chloroplasts arose from cyanobacteria, however, during endosymbiosis, the photosynthetic genes in chloroplasts have been reduced. How these changes occurred during plant evolution was the focu...It has now believed that chloroplasts arose from cyanobacteria, however, during endosymbiosis, the photosynthetic genes in chloroplasts have been reduced. How these changes occurred during plant evolution was the focus of the present study. Beginning with photosystem I （PSI） genes, a homologous comparison of amino acid sequences of 18 subunits of PSI from 10 species of cyanobacteria, chloroplasts in 12 species of eucaryotic algae, and 28 species of plants （including bryophytes, pteridophytes, gymnospermae, dicotyledon and monocotyledon） was undertaken. The data showed that 18 genes of PSI can be divided into two groups： Part Ⅰ including seven genes （psaA, psaB, psaC, psal, psaJ, ycf3 and ycf4） shared both by cyanobacteria and plant chloroplasts; Part Ⅱ containing another 11 genes （psaD, psaE, psaF, psaK, psaL, psaM, btpA, ycf37, psaG, psaH and psaN） appeared to have diversified in different plant groups. Among Part Ⅰ genes, psaC, psaA and psaB had higher homology in all species of cyanobacteria and chloroplasts. Among Part Ⅱ genes, only psaG, psaH and psaN emerged in seed plants.展开更多
Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84%...Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification.展开更多
Objective To compare the effects of autotransfusion and homologous transfusion on milieu interieur and secretion of erythropoietin (EPO). Methods Thirty patients aged 8-21 yr, undergoing surgical correction of kyphosc...Objective To compare the effects of autotransfusion and homologous transfusion on milieu interieur and secretion of erythropoietin (EPO). Methods Thirty patients aged 8-21 yr, undergoing surgical correction of kyphoscoliosis were randomly divided into two groups of fifteen:group A and group B. In group A patients received homologous transfusion during operation, while in group B shed blood was collected from operative field and after filtration RBCs were separated by a differential centrifugation and washed in normal saline and reinfused. Anesthesia was induced with fentanyl 2 μg· kg-1 ,propofol 1 mg·kg-1 and vecuronium 0.1 mg·kg-1 and maintained with inhalation of isoflurane and 50 % N2O in oxygen and intermittent iv boluses of vecuronium and fentanyl. Blood routine, blood gases, electrolytes, plasma osmotic pressure and activated clotting time (ACT) were measured before operation, 2 h after skin incision and at the end of surgery. Blood EPO concentration was measured before operation,at the end展开更多
TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published...TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.展开更多
基金Supported by the Natural Science Foundation of Shanxi Province(No.20031042).
文摘A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.
基金Tianjin Municipal Science and Technology Commission(043182711)
文摘It has now believed that chloroplasts arose from cyanobacteria, however, during endosymbiosis, the photosynthetic genes in chloroplasts have been reduced. How these changes occurred during plant evolution was the focus of the present study. Beginning with photosystem I （PSI） genes, a homologous comparison of amino acid sequences of 18 subunits of PSI from 10 species of cyanobacteria, chloroplasts in 12 species of eucaryotic algae, and 28 species of plants （including bryophytes, pteridophytes, gymnospermae, dicotyledon and monocotyledon） was undertaken. The data showed that 18 genes of PSI can be divided into two groups： Part Ⅰ including seven genes （psaA, psaB, psaC, psal, psaJ, ycf3 and ycf4） shared both by cyanobacteria and plant chloroplasts; Part Ⅱ containing another 11 genes （psaD, psaE, psaF, psaK, psaL, psaM, btpA, ycf37, psaG, psaH and psaN） appeared to have diversified in different plant groups. Among Part Ⅰ genes, psaC, psaA and psaB had higher homology in all species of cyanobacteria and chloroplasts. Among Part Ⅱ genes, only psaG, psaH and psaN emerged in seed plants.
文摘Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification.
文摘Objective To compare the effects of autotransfusion and homologous transfusion on milieu interieur and secretion of erythropoietin (EPO). Methods Thirty patients aged 8-21 yr, undergoing surgical correction of kyphoscoliosis were randomly divided into two groups of fifteen:group A and group B. In group A patients received homologous transfusion during operation, while in group B shed blood was collected from operative field and after filtration RBCs were separated by a differential centrifugation and washed in normal saline and reinfused. Anesthesia was induced with fentanyl 2 μg· kg-1 ,propofol 1 mg·kg-1 and vecuronium 0.1 mg·kg-1 and maintained with inhalation of isoflurane and 50 % N2O in oxygen and intermittent iv boluses of vecuronium and fentanyl. Blood routine, blood gases, electrolytes, plasma osmotic pressure and activated clotting time (ACT) were measured before operation, 2 h after skin incision and at the end of surgery. Blood EPO concentration was measured before operation,at the end
文摘TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.
