Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr Ⅱ-EcoRI DNA fragment with intact HCMV pci gene coding sequence was engineere...Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr Ⅱ-EcoRI DNA fragment with intact HCMV pci gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pci gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pci gene. In fection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 108 cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3'-5'and 5'-3' exonuclease activities.This enzyme is also sensitive to phosphono acetate.展开更多
The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) ...The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) promoter in Bacillus subtilis 168 at 20℃.The yield was 7.5 mg/L,and 90.6%purity was achieved using ammonium sulfate precipitation,Ni–NTA and molecular exclusion.The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109(DE3)and Pseudomonas aeruginosa CGMCC 1.6740,and 48.4%of growth of Staphylococcus aureus CGMCC 1.282,the result is similar to that of 200 ng/mL N-lobe.The minimum inhibitory concentrations of C-lobe were 4,8 and 16 mg/mL,while those of N-lobe were 128,256 and 512μg/mL for E.coli,P.aeruginosa and S.aureus,respectively.This is the frst report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N-and C-lobes in a food-safe strain of B.subtilis.Our fndings ofer the potential to study the structure–function relationship of the N-and C-lobes recombinantly produced in the same host.展开更多
文摘Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr Ⅱ-EcoRI DNA fragment with intact HCMV pci gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pci gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pci gene. In fection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 108 cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3'-5'and 5'-3' exonuclease activities.This enzyme is also sensitive to phosphono acetate.
基金This work was supported by the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(31970045)+2 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difcult.Here,we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P_(veg) promoter in Bacillus subtilis 168 at 20℃.The yield was 7.5 mg/L,and 90.6%purity was achieved using ammonium sulfate precipitation,Ni–NTA and molecular exclusion.The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109(DE3)and Pseudomonas aeruginosa CGMCC 1.6740,and 48.4%of growth of Staphylococcus aureus CGMCC 1.282,the result is similar to that of 200 ng/mL N-lobe.The minimum inhibitory concentrations of C-lobe were 4,8 and 16 mg/mL,while those of N-lobe were 128,256 and 512μg/mL for E.coli,P.aeruginosa and S.aureus,respectively.This is the frst report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N-and C-lobes in a food-safe strain of B.subtilis.Our fndings ofer the potential to study the structure–function relationship of the N-and C-lobes recombinantly produced in the same host.
