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
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.
