AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunorea...AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.展开更多
Human extracellular superoxide dismutase(hEC-SOD) is a secreted tetrameric protein involved in the protection of a human body from oxygen free radicals. Its three-dimensional structure has not been confirmed. hEC-SOD ...Human extracellular superoxide dismutase(hEC-SOD) is a secreted tetrameric protein involved in the protection of a human body from oxygen free radicals. Its three-dimensional structure has not been confirmed. hEC-SOD couldn′t be expressed in E.coli. We constructed a hybrid enzyme, which comprises the N-terminal and C-terminal domains from hEC-SOD, fused it to human Cu,Zn-SOD. The hybrid enzyme is expressed successfully in E.coli. Further, we analyzed the expression of hEC-SOD in E.coli by mRNA differential displaying.展开更多
A novel oligosaccharide was isolated and purified from the mucus of the loach, Misgurnus anguillicaudatus. It was identified by several qualitative tests and characterized by elementary analysis, UV and IR spectrum. ...A novel oligosaccharide was isolated and purified from the mucus of the loach, Misgurnus anguillicaudatus. It was identified by several qualitative tests and characterized by elementary analysis, UV and IR spectrum. Its average molecular weight (Mw=1539.4) was determined by gel permeation chromatography. The major structural monomers of Misgurnus anguillicaudatus oligosaccharide were identified to be D-galactose and L-fucose by paper chromatography and gas chromatography.展开更多
To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetram...To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.展开更多
Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20 μg g -1 net cells, and the band inten...Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20 μg g -1 net cells, and the band intensity ratio of 28 S/ 18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.展开更多
Auxin response factors (ARFs) play a central role in plants as transcriptional factors in response to auxin. The Arabidopsis ARF8 gene is a light-inducible gene and ARF8 protein might control auxin homeostasis in a ne...Auxin response factors (ARFs) play a central role in plants as transcriptional factors in response to auxin. The Arabidopsis ARF8 gene is a light-inducible gene and ARF8 protein might control auxin homeostasis in a negative feed-back fashion through regulation of GH3 gene expression. In a double mutant designated infertile line including arf8-1 (a T-DNA insertion mutant of ARF8), we isolated fertility1-1 (fer1-1), a mutant of Fer1, which acts synergistically with ARF8 to control the development of the anther and filament in Arabidopsis. Genetics analysis has demonstrated that fer 1-1 is a T-DNA insertion line, indicating that Fer1 might be cloned by inverse polymerase chain reaction (PCR) or the TAIL-PCR approach. Phenotypic identification and molecular analysis of fer 1-1 and the infertile line will be helpful to characterize the function of Fer1, to further study the function of ARF8, and to reveal the molecular mechanism underlying the interaction of Fer1 and ARF8 in controlling development of the anther and filament.展开更多
基金Supported by the Medical Science Foundation for Distinguished Scholars of Henan Province, No. 200084
文摘AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.
文摘Human extracellular superoxide dismutase(hEC-SOD) is a secreted tetrameric protein involved in the protection of a human body from oxygen free radicals. Its three-dimensional structure has not been confirmed. hEC-SOD couldn′t be expressed in E.coli. We constructed a hybrid enzyme, which comprises the N-terminal and C-terminal domains from hEC-SOD, fused it to human Cu,Zn-SOD. The hybrid enzyme is expressed successfully in E.coli. Further, we analyzed the expression of hEC-SOD in E.coli by mRNA differential displaying.
文摘A novel oligosaccharide was isolated and purified from the mucus of the loach, Misgurnus anguillicaudatus. It was identified by several qualitative tests and characterized by elementary analysis, UV and IR spectrum. Its average molecular weight (Mw=1539.4) was determined by gel permeation chromatography. The major structural monomers of Misgurnus anguillicaudatus oligosaccharide were identified to be D-galactose and L-fucose by paper chromatography and gas chromatography.
文摘To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.
文摘Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20 μg g -1 net cells, and the band intensity ratio of 28 S/ 18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.
文摘Auxin response factors (ARFs) play a central role in plants as transcriptional factors in response to auxin. The Arabidopsis ARF8 gene is a light-inducible gene and ARF8 protein might control auxin homeostasis in a negative feed-back fashion through regulation of GH3 gene expression. In a double mutant designated infertile line including arf8-1 (a T-DNA insertion mutant of ARF8), we isolated fertility1-1 (fer1-1), a mutant of Fer1, which acts synergistically with ARF8 to control the development of the anther and filament in Arabidopsis. Genetics analysis has demonstrated that fer 1-1 is a T-DNA insertion line, indicating that Fer1 might be cloned by inverse polymerase chain reaction (PCR) or the TAIL-PCR approach. Phenotypic identification and molecular analysis of fer 1-1 and the infertile line will be helpful to characterize the function of Fer1, to further study the function of ARF8, and to reveal the molecular mechanism underlying the interaction of Fer1 and ARF8 in controlling development of the anther and filament.
